Engineered heme-binding compositions and uses thereof

ABSTRACT

Described herein are heme-binding compositions and methods relating to their use, for example methods of treatment of sepsis and rhabdomyolysis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. § 120 of U.S.application Ser. No. 15/968,116 filed May 1, 2018, which issued as U.S.Pat. No. 10,501,729 on Dec. 10, 2019, which is a continuation under 35U.S.C. § 120 of U.S. application Ser. No. 14/892,252 filed Nov. 19,2015, which issued as U.S. Pat. No. 9,988,617 on Jun. 5, 2018, which isa 35 U.S.C. § 371 National Phase Entry Application of InternationalApplication No. PCT/US2014/038945 filed May 21, 2014, which designatesthe U.S., and which claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/825,707 filed May 21, 2013, the contentsof which are incorporated herein by reference in their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos.N66001-11-1-4180 awarded by the U.S. Department of Defense. Thegovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 18, 2015, isnamed 002806-076882-US_SL.txt and is 51,193 bytes in size.

TECHNICAL FIELD

The technology described herein relates to methods and compositionsrelating to the treatment of heme and/or myoglobin-associated diseaseand disorders, e.g. sepsis, rhabdomyolysis, crush injury, and the like.

BACKGROUND

Sepsis is a lethal condition that is often associated with a seriousmicrobial infection. However, while many hypotheses have been putforward, the exact cause of septic shock is not agreed upon andtherapeutics based on targeting the source of these various hypotheseshave generally failed in (or prior to) clinical trials. The currenttreatment generally includes administration of antibiotics. Pastclinical trials have focused on limiting the immune systems response tomicrobial infections, thereby reducing the “Cytokine Storm” that hasbeen hypothesized to be the causative agent of sepsis. In addition,people have looked to use dialysis to remove cytokines.

SUMMARY

Described herein are methods and compositions relating to the treatmentof heme and/or myoglobin-associated disease and disorders, e.g. sepsisand rhabdomyolysis. The technology described herein is based upon therecognition that excess free heme in the blood can play a role in theprogression of sepsis. In a septic patient or animal, microbialinfections can lead to a large increase in Red Blood Cell (RBS) lysis,which in turn leads to a significant increase in soluble free heme inthe blood stream. This increase overwhelms the endogenous levels ofhemopexin, which normally scavenges endogenous levels of heme, leadingto dangerously high levels of heme. Excess heme in the blood providesmicrobial pathogens with a readily available source of iron, which canbe limiting agent in microbial growth and hemoglobin and heme maysubstantially contribute to microbe-induced inflammation when bacterialor viral infection coexists with blood.

As demonstrated herein, hemopexin fusion proteins can be used to lowerthe level of free heme in the blood of a subject, e.g. to treat sepsis.In one aspect, described herein is a heme-binding molecule and/orcomposition comprising a hemopexin domain conjugated to a Fc domain. Insome embodiments, the hemopexin domain is a polypeptide comprising thesequence of SEQ ID NO: 2. In some embodiments, the hemopexin domain is apolypeptide having the sequence of SEQ ID NO: 2. In some embodiments,the composition has the sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

In one aspect, described herein is a method of reducing the level offree heme in the blood of a subject, the method comprising contactingthe blood of the subject with a heme-binding molecule and/or compositiondescribed herein. In some embodiments, the method further comprisesremoving a portion of the subject's blood prior to the contacting stepand performing the contacting step extracorporeally and then returningthe portion of the subject's blood to the subject. In some embodiments,the heme-binding molecule and/or composition is bound to a solidsubstrate of an extracorporeal device. In some embodiments, the solidsubstrate is a filter or affinity column. In some embodiments, theheme-binding molecule and/or composition can be administered to asubject as a therapeutic agent.

In one aspect, described herein is a method of producing a heme-bindingmolecule and/or composition, the method comprising culturing a cellcomprising a nucleic acid encoding a heme-binding molecule and/orcomposition described herein under conditions suitable for theproduction of proteins and purifying the heme-binding molecule and/orcomposition by affinity purification with a stabilization domain bindingreagent. In some embodiments, the cell is a microbial cell or amammalian cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an image of an SDS gel showing the purity of isolatedFc-Hemopexin fusions.

FIG. 2 depicts a graph of Fc-Hemopexin and Fc-Hemopexin-NT binding tofree hemin.

FIG. 3 depicts a graph of the heme binding of Fc fusions with variantsof the N-terminal domain of Hemopexin.

FIG. 4 depicts a graph of heme binding of Fc Fusions with variants ofFull Length Hemopexin was also determined.

FIG. 5 depicts a graph of FcHemopexin variants binding to myoglobin.

DETAILED DESCRIPTION

As described herein, the inventors have discovered that certainhemopexin fusion proteins can be used to binding free heme in blood.Accordingly, provided herein are methods and compositions relating tothese fusion proteins and their use for reducing heme levels in theblood, e.g. for the treatment of sepsis.

In one aspect, the invention described herein relates to a heme-bindingmolecule and/or composition comprising a hemopexin domain conjugated toa Fc domain. In some embodiments, the composition can be a multimer. Asused herein, “hemopexin domain” refers to a domain or portion of apolypeptide composition described herein comprising a hemopexinpolypeptide or a fragment thereof “Hemopexin” (also referred to as“haemopexin,” “HPX,” or “beta-1B-glycoprotein” refers to a protein withthe highest known affinity for heme and which interacts with the LRP1receptor when complexed with heme. The sequences of hemopexin for avariety of species are known, e.g. human hemopexin (NCBI Gene ID: 3263(SEQ ID NO: 1; NCBI Ref Seq: NP_00604; polypeptide)(SEQ ID NO: 6; NCBIRef Seq: NM_000613; mRNA).

A hemopexin polypeptide can comprise SEQ ID NO: 1 or a homolog, variant,and/or functional fragment thereof. In some embodiments, a hemopexinpolypeptide can comprise amino acids 24 to 462 of SEQ ID NO: 1 (i.e. themature hemopexin polypeptide with the signal peptide sequence removed),or a homolog, variant, and/or functional fragment thereof. In someembodiments, a hemopexin domain can comprise amino acid 24 to amino acid256 of SEQ ID NO: 1 or a homolog, variant, and/or functional fragmentthereof. In some embodiments, a hemopexin domain can comprise amino acid27 to amino acid 213 of SEQ ID NO: 1 or a homolog, variant, and/orfunctional fragment thereof. In some embodiments, a hemopexin domain cancomprise amino acid 1 to amino acid 213, 220, 233, or 256 of SEQ ID NO:1 or a homolog, variant, and/or functional fragment thereof. In someembodiments, a hemopexin domain can comprise amino acid 24 to amino acid213, 220, 233, or 256 of SEQ ID NO: 1 or a homolog, variant, and/orfunctional fragment thereof. In some embodiments, a hemopexin domain cancomprise amino acid 27 to amino acid 213, 220, 233, or 256 of SEQ ID NO:1 or a homolog, variant, and/or functional fragment thereof. In someembodiments, a hemopexin polypeptide as described herein can be ahomolog, derivative, variant, conservative substitution variant,deletion mutant, insertion mutant, or functional fragment of the aminoacid sequences described above herein. In some embodiments, a hemopexindomain can comprise a mutation wherein the residues corresponding toresidues 220-226 of SEQ ID NO: 1 have been replaced with the sequenceGSGS (SEQ ID NO: 18).

In some embodiments, a hemopexin domain can comprise amino acid 24 toamino acid 256 of SEQ ID NO: 2 or a homolog, variant, and/or functionalfragment thereof. In some embodiments, a hemopexin domain can compriseamino acid 27 to amino acid 213 of SEQ ID NO: 2 or a homolog, variant,and/or functional fragment thereof. In some embodiments, a hemopexindomain can comprise amino acid 1 to amino acid 213, 220, 233, or 256 ofSEQ ID NO: 2 or a homolog, variant, and/or functional fragment thereof.In some embodiments, a hemopexin domain can comprise amino acid 24 toamino acid 213, 220, 233, or 256 of SEQ ID NO: 2 or a homolog, variant,and/or functional fragment thereof. In some embodiments, a hemopexindomain can comprise amino acid 27 to amino acid 213, 220, 233, or 256 ofSEQ ID NO: 2 or a homolog, variant, and/or functional fragment thereof.In some embodiments, a hemopexin polypeptide as described herein can bea homolog, derivative, variant, conservative substitution variant,deletion mutant, insertion mutant, or functional fragment of the aminoacid sequences described above herein. In some embodiments, a hemopexindomain can comprise a mutation wherein the residues corresponding toresidues 220-226 of SEQ ID NO: 2 have been replaced with the sequenceGSGS (SEQ ID NO: 18).

As used herein, a “functional fragment” of, e.g. SEQ ID NO: 1 is afragment or segment of that polypeptide which can bind heme at least 10%as strongly as the reference polypeptide (i.e. SEQ ID NO: 1), e.g. atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 75%, at least 90%, at least 100% as strongly, or more strongly.Assays for determining heme concentrations and binding of a protein toheme are well known in the art and include, by way of non-limitingexample, spectroscopic titrations using dithionite, e.g. as described inAirola et al. Biochemistry 2001 49:43217-4338; the assay described inU.S. Pat. No. 4,340,668; or any of the assays described in Sinclair etal. Current Protocols in Toxicology 2001; unit 8.3. Each of theforegoing references is incorporated by reference herein in itsentirety. A functional fragment can comprise conservative substitutionsof the sequences disclosed herein. In some embodiments, heme binding caninclude myoglobin binding activity.

Variants of the isolated peptides described herein (e.g. SEQ ID NOs:1-5) can be obtained by mutations of native nucleotide or amino acidsequences, for example SEQ ID NO: 1 or a nucleotide sequence encoding apeptide comprising SEQ ID NO:1. A “variant,” as referred to herein, is apolypeptide substantially homologous to an hemopexin polypeptidedescribed herein (e.g. SEQ ID NOs: 1 and 2), but which has an amino acidsequence different from that of one of the sequences described hereinbecause of one or a plurality of deletions, insertions or substitutions.

A homolog of a hemopexin polypeptide as described herein can alsocomprise amino acid sequences that are homologous to the regions ofhemopexin comprised by the hemopexin polypeptide described herein.

The variant amino acid or DNA sequence preferably is at least 60%, atleast 70%, at least 80%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or more, identical to the sequence from whichit is derived (referred to herein as an “original” sequence). The degreeof homology (percent identity) between an original and a mutant sequencecan be determined, for example, by comparing the two sequences usingfreely available computer programs commonly employed for this purpose onthe world wide web. The variant amino acid or DNA sequence preferably isat least 60%, at least 70%, at least 80%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or more, similar to the sequencefrom which it is derived (referred to herein as an “original” sequence).The degree of similarity (percent similarity) between an original and amutant sequence can be determined, for example, by using a similaritymatrix. Similarity matrices are well known in the art and a number oftools for comparing two sequences using similarity matrices are freelyavailable online, e.g. BLASTp (available on the world wide web athttp://blast.ncbi.nlm.nih.gov).

Alterations of the original amino acid sequence can be accomplished byany of a number of known techniques known to one of skill in the art.Mutations can be introduced, for example, at particular loci bysynthesizing oligonucleotides containing a mutant sequence, flanked byrestriction sites enabling ligation to fragments of the native sequence.Following ligation, the resulting reconstructed sequence encodes ananalog having the desired amino acid insertion, substitution, ordeletion. Alternatively, oligonucleotide-directed site-specificmutagenesis procedures can be employed to provide an altered nucleotidesequence having particular codons altered according to the substitution,deletion, or insertion required. Techniques for making such alterationsinclude those disclosed by Walder et al. (Gene 42:133, 1986); Bauer etal. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19);Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press,1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are hereinincorporated by reference in their entireties. In some embodiments, anisolated peptide as described herein can be chemically synthesized andmutations can be incorporated as part of the chemical synthesis process.

Variants can comprise conservatively substituted sequences, meaning thatone or more amino acid residues of an original peptide are replaced bydifferent residues, and that the conservatively substituted peptideretains a desired biological activity, i.e., the ability to bind heme,that is essentially equivalent to that of the original peptide. Examplesof conservative substitutions include substitutions that do not changethe overall or local hydrophobic character, substitutions that do notchange the overall or local charge, substitutions by residues ofequivalent sidechain size, or substitutions by sidechains with similarreactive groups.

A given amino acid can be replaced by a residue having similarphysiochemical characteristics, e.g., substituting one aliphatic residuefor another (such as Ile, Val, Leu, or Ala for one another), orsubstitution of one polar residue for another (such as between Lys andArg; Glu and Asp; or Gln and Asn). Other such conservativesubstitutions, e.g., substitutions of entire regions having similarhydrophobicity characteristics or substitutions of residues with similarsidechain volume are well known. Isolated peptides comprisingconservative amino acid substitutions can be tested in any one of theassays described herein to confirm that a desired activity, e.g. theability to bind heme, is retained, as determined by the assays describedelsewhere herein.

Amino acids can be grouped according to similarities in the propertiesof their side chains (in A. L. Lehninger, in Biochemistry, second ed.,pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A),Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2)uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N),Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His(H). Alternatively, naturally occurring residues can be divided intogroups based on common side-chain properties: (1) hydrophobic:Norleucine, Met, Ala, Val, Leu, Ile, Phe, Trp; (2) neutral hydrophilic:Cys, Ser, Thr, Asn, Gln, Ala, Tyr, His, Pro, Gly; (3) acidic: Asp, Glu;(4) basic: His, Lys, Arg; (5) residues that influence chain orientation:Gly, Pro; (6) aromatic: Trp, Tyr, Phe, Pro, His, or hydroxyproline.Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Particularly preferred conservative substitutions for use in thevariants described herein are as follows: Ala into Gly or into Ser; Arginto Lys; Asn into Gln or into His; Asp into Glu or into Asn; Cys intoSer; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asnor into Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lysinto Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Pheinto Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyror into Phe; Tyr into Phe or into Trp; and/or Phe into Val, into Tyr,into Ile or into Leu. In general, conservative substitutions encompassresidue exchanges with those of similar physicochemical properties (i.e.substitution of a hydrophobic residue for another hydrophobic aminoacid).

Any cysteine residue not involved in maintaining the proper conformationof the isolated peptide as described herein can also be substituted,generally with serine, to improve the oxidative stability of themolecule and prevent aberrant crosslinking. Conversely, cysteine bond(s)can be added to the isolated peptide as described herein to improve itsstability or facilitate multimerization.

In some embodiments, a functional fragment of hemopexin can comprisefrom about amino acid 24 to about amino acid 256 of SEQ ID NO: 1. Insome embodiments, a functional fragment of hemopexin comprise amino acid24 to amino acid 256 of SEQ ID NO: 1, i.e. SEQ ID NO: 2. In someembodiments, a functional fragment of hemopexin can be a polypeptidehaving the sequence of SEQ ID NO: 2.

As used herein, “a Fc domain” refers to domain, part, or portion of apolypeptide comprising an Fc polypeptide. As used herein, a “Fcpolypeptide” refers to the region of an antibody that interacts with Fcreceptors and certain components of the complement system. The Fc regionfor a given type of antibody will be constant for all antibodies of thattype in an individual, whereas the Fab region of the antibody will vary,providing antigen specificity. In some embodiments, a Fc polypeptide canbe a polypeptide having the sequence of SEQ ID NO: 7 or a or a homolog,variant, and/or functional fragment thereof. In some embodiments, a Fcpolypeptide can be a polypeptide having the sequence of SEQ ID NO: 8 ora or a homolog, variant, and/or functional fragment thereof. In someembodiments, a Fc polypeptide can be a polypeptide having the sequenceof SEQ ID NO: 17 or a or a homolog, variant, and/or functional fragmentthereof. In the context of a FC polypeptide, a functional fragment is afragment or segment of that polypeptide which can bind or be bound by Fcreceptors and/or C1q at least 10% as strongly as the referencepolypeptide (i.e. SEQ ID NO: 7), e.g. at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 75%, at least 90%, atleast 100% as strongly, or more strongly. Assays for the binding of aligand and its receptor are well known in the art.

In such embodiments, the Fc region can comprise at least one regionselected from the group consisting of a hinge region, a CH2 region, aCH3 region, and any combinations thereof. By way of example, in someembodiments, a CH2 region can be excluded from the portion of the Fcregion as the second domain. In one embodiment, Fc region comprisedcomprises a hinge region, a CH2 domain and a CH3 domain.

In some embodiments, the Fc region can be can be used to facilitateexpression and purification of the engineered molecules and compositionsdescribed herein. The N terminal Fc has been shown to improve expressionlevels, protein folding and secretion of the fusion partner. Inaddition, the Fc has a staphylococcal Protein A binding site, which canbe used for one-step purification protein A affinity chromatography. SeeLo K M et al. (1998) Protein Eng. 11: 495-500. Further, the Protein Abinding site can be used to facilitate binding of Protein A-expressingor Protein G-expressing microbes in the absence of calcium ions.Further, such Fc regions have a molecule weight above a renal thresholdof about 45 kDa, thus reducing the possibility of engineered moleculesbeing removed by glomerular filtration. Additionally, the Fc region canallow dimerization of two engineered heme-binding domain molecules toform a multimeric complex, such as a dimer.

In some embodiments, an Fc region or a fragment thereof can comprise atleast one mutation, e.g., to modify the performance of the engineeredheme-binding molecules and/or compositions. For example, in someembodiments, a half-life of the engineered heme-binding molecules and/orcompositions described herein can be increased, e.g., by mutating anamino acid lysine (K) at the residue 224 of SEQ ID NO: 5 to alanine (A).Other mutations, e.g., located at the interface between the CH2 and CH3domains shown in Hinton et al (2004) J Biol Chem. 279:6213-6216 andVaccaro C. et al. (2005) Nat Biotechnol. 23: 1283-1288, can be also usedto increase the half-life of the IgG1 and thus the engineeredheme-binding molecules and/or compositions.

In some embodiments, the Fc polypeptide can comprise a N297D mutation,which results in an aglycosylated Fc polypeptide.

In some embodiments, the Fc polypeptide is a polypeptide that can bebound by an Fc receptor and internalized into a cell, e.g. intosubcellular compartments. This can remove the bound heme from the bloodand direct it into cellular recycling pathways. The Heme-Hemopexincomplex is typically removed form the blood stream by CD91 mediatedendocytosis. The Fc-Hemopexin molecule can increase this clearance rateby taking advantage of endocytosis and recycling of Fc containingproteins via its interaction with Fc receptors.

In one aspect, described herein is an engineered heme-binding moleculecomprising a hemopexin domain and a linker, substrate-binding domain,and/or microbe-binding molecule conjugated thereto. As used herein,“engineered” refers to the aspect of having been manipulated by the handof man. For example, a polynucleotide is considered to be “engineered”when two or more sequences, that are not linked together in that orderin nature, are manipulated by the hand of man to be directly linked toone another in the engineered polynucleotide. For example, in someembodiments of the present invention, an engineered molecule comprisesmultiple domains that are each found in nature, but are not found in thesame transcript in nature. As is common practice and is understood bythose in the art, progeny and copies of an engineered polynucleotide aretypically still referred to as “engineered” even though the actualmanipulation was performed on a prior entity.

Multiple domains of the heme-binding composition and/or molecule can belinked together by a linker. Further, the heme-binding compositionand/or molecule can be conjugated to a carrier scaffold via linker.Accordingly, as used in this disclosure, the term “linker” means amoiety that connects two parts of a compound or molecule. Linkerstypically comprise a direct bond or an atom such as oxygen or sulfur, aunit such as NR¹, C(O), C(O)O, OC(O)O, C(O)NH, NHC(O)O, NH, SS, SO, SO₂,SO₃, and SO₂NH, or a chain of atoms, such as substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, where one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, NH, C(O)N(R¹)₂, C(O), cleavable linkinggroup, substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocyclic; where R¹ ishydrogen, acyl, aliphatic or substituted aliphatic. In some embodiments,the linker can be a non-covalent association (e.g., by non-covalentinteractins) of the two parts of a molecule being conjugated together.Some exemplary non-covalent on ionic interactions, van der Waalsinteractions, dipole-dipole interactions, hydrogen bonds, electrostaticinteractions, and/or shape recognition interactions.

In some embodiments, the linker can comprise at least one cleavablelinking group. A cleavable linking group is one which is sufficientlystable under one set of conditions, but which is cleaved under adifferent set of conditions to release the two parts the linker isholding together. In some embodiments, the cleavable linking group iscleaved at least 10 times or more, e.g., at least 100 times faster undera first reference condition (which can, e.g., be selected to mimic orrepresent a microbe-infected condition, such as a microbe-infectedtissue or body fluid, or a microbial biofilm occurring in anenvironment) than under a second reference condition (which can, e.g.,be selected to mimic or represent non-infected conditions, e.g., foundin the non-infected blood or serum, or in an non-infected environment).

Cleavable linking groups are susceptible to cleavage agents, e.g.,hydrolysis, pH, redox potential or the presence of degradativemolecules. Generally, cleavage agents are more prevalent or found athigher levels or activities at a site of interest (e.g. a microbialinfection) than in non-infected area. Examples of such degradativeagents include: redox agents which are selected for particularsubstrates or which have no substrate specificity, including, e.g.,oxidative or reductive enzymes or reductive agents such as mercaptans,present in cells, that can degrade a redox cleavable linking group byreduction; esterases; amidases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific)and proteases, and phosphatases.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell, organ, or tissue to be targeted. Insome embodiments, cleavable linking group is cleaved at least 1.25, 1.5,1.75, 2, 3, 4, 5, 10, 25, 50, or 100 times faster under a firstreference condition (or under in vitro conditions selected to mimic amicrobe-infected condition, such as a microbe-infected tissue or bodyfluid, or a microbial biofilm occurring in an environment or on aworking surface) than under a second reference condition (or under invitro conditions selected to mimic non-infected conditions, e.g., foundin the non-infected blood or serum, or in an non-infected environment).In some embodiments, the cleavable linking group is cleaved by less than90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% in thenon-infected conditions, e.g., found in the non-infected blood or serum,or in an non-infected environment, as compared to a microbe-infectedcondition, such as a microbe-infected tissue or body fluid, or amicrobial biofilm occurring in an environment or on a working surface.

Exemplary cleavable linking groups include, but are not limited to,hydrolyzable linkers, redox cleavable linking groups (e.g., —S—S— and—C(R)₂—S—S—, wherein R is H or C₁-C₆ alkyl and at least one R is C₁-C₆alkyl such as CH₃ or CH₂CH₃); phosphate-based cleavable linking groups(e.g., —O—P(O)(OR)—O—, —O—P(S)(OR)—O—, —O—P(S)(SR)—O—, —S—P(O)(OR)—O—,—O—P(O)(OR)—S—, —S—P(O)(OR)—S—, —O—P(S)(ORk)-S—, —S—P(S)(OR)—O—,—O—P(O)(R)—O—, —O—P(S)(R)—O—, —S—P(O)(R)—O—, —S—P(S)(R)—O—,—S—P(O)(R)—S—, —O—P(S)(R)—S—, —O—P(O)(OH)—O—, —O—P(S)(OH)—O—,—O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—,—O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—,—S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, and —O—P(S)(H)—S—, whereinR is optionally substituted linear or branched C₁-C₁₀ alkyl); acidcelavable linking groups (e.g., hydrazones, esters, and esters of aminoacids, —C≡NN— and —OC(O)—); ester-based cleavable linking groups (e.g.,—C(O)O—); peptide-based cleavable linking groups, (e.g., linking groupsthat are cleaved by enzymes such as peptidases and proteases in cells,e.g., —NHCHR^(A)C(O)NHCHR^(B)C(O)—, where R^(A) and R^(B) are the Rgroups of the two adjacent amino acids). A peptide based cleavablelinking group comprises two or more amino acids. In some embodiments,the peptide-based cleavage linkage comprises the amino acid sequencethat is the substrate for a peptidase or a protease. In someembodiments, an acid cleavable linking group is cleavable in an acidicenvironment with a pH of about 6.5 or lower (e.g., about 6.5, 6.0, 5.5,5.0, or lower), or by agents such as enzymes that can act as a generalacid.

Without limitations, the linker can be selected to provide a desiredfunction or property to the heme-binding molecules and/or compositionsdisclosed herein. For example, the linker can be selected or configuredaccording to a specific need or use of the heme-binding molecules and/orcompositions. By way of example only, in some embodiments, linker can beselected or configured to have a sufficient length and flexibility suchthat it can allow for the microbe-binding domain to orient in a desiredorientation with respect to a microbe. In some embodiments, the linkercan be selected or configured to allow multimerization of at least twoengineered heme-binding molecules and/or compositions (e.g., to from adi-, tri-, tetra-, penta-, hexa- or higher multimeric complex) whileretaining biological activity (e.g., heme-binding activity). In someembodiments, the linker can be selected or configured to inteact with asecond domain (e.g. an Fc domain) to allow multimerization of at leasttwo engineered heme-binding molecules and/or compositions (e.g., to froma di-, tri-, tetra-, penta-, hexa- or higher multimeric complex) whileretaining heme-binding activity.

In some embodiments, the linker can be selected or configured tofacilitate expression and purification of the engineered heme-bindingmolecules and/or compositions described herein. In some embodiments, thelinker can be selected or configured to provide a recognition site for aprotease or a nuclease. In addition, the linker can be non-reactive withthe functional components of the engineered molecule described herein.For example, minimal hydrophobic or charged character to react with adomain of the heme-binding molecule and/or composition. In someembodiments, the linker can be part of a domain of the heme-bindingmolecule and/or composition.

In some embodiments, the linker can be a peptide or a nucleic acid. Insome embodiments, the peptide linker can vary from about 1 to about 1000amino acids long, from about 10 to about 500 amino acids long, fromabout 30 to about 300 amino acids long, or from about 50 to about 150amino acids long. In some embodiments, the peptidyl linker is from about1 amino acid to about 20 amino acids long. In some embodiments, thenucleic acid linker can vary from about 1 to about 1000 nucleotideslong, from about 10 to about 500 nucleotides long, from about 30 toabout 300 nucleotides, or from about 50 to about 150 nucleotides. Longeror shorter linker sequences can be also used for the engineeredheme-binding molecules and/or compositions described herein.

The peptidyl linker can be configured to have a sequence comprising atleast one of the amino acids selected from the group consisting ofglycine (Gly), serine (Ser), asparagine (Asn), threonine (Thr),methionine (Met) or alanine (Ala). Such amino acids are generally usedto provide flexibility of a linker. However, in some embodiments, otheruncharged polar amino acids (e.g., Gln, Cys or Tyr), nonpolar aminoacids (e.g., Val, Leu, Ile, Pro, Phe, and Trp). In alternativeembodiments, polar amino acids can be added to modulate the flexibilityof a linker. One of skill in the art can control flexibility of a linkerby varying the types and numbers of residues in the linker. See, e.g.,Perham, 30 Biochem. 8501 (1991); Wriggers et al., 80 Biopolymers 736(2005).

In some embodiments, the peptidyl linker can comprise form 1 to about 25amino acids, i.e., one, two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three,twenty-four, or twenty-five amino acids. In some embodiments, thepeptidyl linker linking the first and second domain comprises the aminoacid sequence HHHHHH (SEQ ID NO: 34).

In some embodiments, when the heme-binding molecules and/or compositionscomprise an Fc region, the linker linking the heme binding and the Fcdomain is not a bond or a peptide.

In some embodiments, the linker is a bond.

In some embodiments, the linker conjugating a heme-binding moleculeand/or composition to a carrier scaffold is a polyethylene glycol.Exemplary PEGs for use as linkers include, but are not limited to,PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K, and thelike.

In some embodiments, the linker can be albumin, transferrin or afragment thereof. Without limitations, such linkers can be used toextend the plasma half-life of the engineered heme-binding moleculesand/or compositions. Thus, engineered heme-binding molecules and/orcompositions can be useful for in vivo administration. See Schmidt S R(2009) Curr Opin Drug Discov Devel. 12: 284. In some embodiments, thelinker can be a physical substrate, e.g., microparticles or magneticmicrobes.

A linker between a first domain and a second domain can providesufficient distance between the first and the second domain to allow thefirst domain to interact with heme. Accordingly, in some embodiments,the distance between the first domain and the second domain can rangefrom about 50 angstroms to about 5000 angstroms, from about 100angstroms to about 2500 angstroms, or from about 200 angstroms to about1000 angstroms.

The linkers can be of any shape. For example, the linker can be linear,folded, branched. In some embodiments, the linker can adopt the shape ofa carrier scaffold. In some embodiments, the linkers can be linear. Insome embodiments, the linkers can be folded. In some embodiments, thelinkers can be branched. For branched linkers, each branch of a moleculecan comprise at least one heme-binding domain. In other embodiments, thelinker adopts the shape of the physical substrate.

In some embodiments, the heme-binding molecules and/or compositions cancomprise a functional group for conjugating the hemopexin domain toanother molecule, a composition, a physical substrate, and the like. Forexample, a second domain can comprise a functional group for covalentlylinking the heme-binding domain with another molecule molecule, acomposition, a physical substrate, or the like. Some exemplaryfunctional groups for conjugation include, but are not limited to, anamino group, a N-substituted amino group, a carboxyl group, a carbonylgroup, an acid anhydride group, an aldehyde group, a hydroxyl group, anepoxy group, a thiol, a disulfide group, an alkenyl group, a hydrazinegroup, a hydrazide group, a semicarbazide group, a thiosemicarbazidegroup, one partner of a binding pair, an amide group, an aryl group, anester group, an ether group, a glycidyl group, a halo group, a hydridegroup, an isocyanate group, an urea group, an urethane group, and anycombinations thereof.

In some embodiments, the heme-binding molecules and/or compositionsdisclosed herein can be immobilized on a carrier scaffold for a varietyof applications or purposes. For example, the engineered heme-bindingmolecules and/or compositions can be immobilized on a carrier scaffoldfor easy handling during usage, e.g., for isolation, observation ormicroscopic imaging.

The attachment of the heme-binding molecules and/or compositionsdisclosed herein to a surface of the carrier scaffold can be performedwith multiple approaches, for example, by direct cross-linking theengineered heme-binding molecules and/or compositions to the carrierscaffold surface; cross-linking the engineered heme-binding molecule tothe carrier scaffold surface via a nucleic acid matrix (e.g., DNA matrixor DNA/oligonucleotide origami structures) for orientation andconcentration to increase detection sensitivity; cross-linking theheme-binding molecules and/or compositions to the carrier scaffoldsurface via a dendrimer-like structure (e.g., PEG/Chitin-structure) toincrease detection sensitivity; attracting heme-binding molecules and/orcompositions coated magnetic microbeads to the carrier scaffold surfacewith a focused magnetic field gradient applied to the carrier scaffoldsurface, attaching an engineered heme-binding molecules and/orcompositions to a carrier scaffold via biotin-avidin orbiotin-avidin-like interaction, or any other art-recognized methods.

Without limitations, any conjugation chemistry known in the art forconjugating two molecules or different parts of a composition togethercan be used for conjugating at least one engineered heme-bindingmolecules and/or compositions to a carrier scaffold. Exemplary couplingmolecules and/or functional groups for conjugating at least oneengineered heme-binding molecules and/or compositions to a substrateinclude, but are not limited to, a polyethylene glycol (PEG,NH2-PEG_(X)-COOH which can have a PEG spacer arm of various lengths X,where 1<X<100, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K,PEG-40K, and the like), maleimide conjugation agent, PASylation,HESylation, Bis(sulfosuccinimidyl) suberate conjugation agent, DNAconjugation agent, peptide conjugation agent, silane conjugation agent,polysaccharide conjugation agent, hydrolyzable conjugation agent, andany combinations thereof.

For engineered heme-binding molecules and/or compositions to beimmobilized on or conjugated to a carrier scaffold, the heme-bindingmolecules and/or compositions described herein can further comprise atleast one (e.g., one, two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,eighteen, nineteen, twenty or more) second domain, e.g., adapted fororienting the heme-binding domain away from the carrier scaffoldsurface. In some embodiments, the carrier scaffold surface can befunctionalized with a coupling molecule to facilitate the conjugation ofengineered heme-binding molecules and/or compositions to the solidsurface.

Accordingly, in some embodiments, the second domain can be selected orconfigured to provide one or more functional groups for conjugating theheme-binding domain with a carrier scaffold or a deteactable label. Adomain adapted for conjugating the heme-binding molecule to a carrierscaffold is also referred to as a “conjugation domain” herein. As usedherein, the term “conjugation domain” refers to any molecule or portionthereof that facilitates the conjugation of the engineered moleculesdescribed herein to a carrier scaffold.

In some embodiments, length of the conjugation domain can vary from 1amino acid residue to about 10 amino acid residues, or about 2 aminoacid residues to about 5 amino acid residues. Determination of anappropriate amino acid sequence of the oconjugatio domain for bindingwith different carrier scaffolds is well within one of skill in the art.For example, according to one or more embodiments, the conjugationdomain can comprise an amino acid sequence of AKT (SEQ ID NO: 35), whichprovides a single biotinylation site for subsequent binding tostreptavidin. Preferably the AKT is at the terminus or near the terminus(e.g., within less than 10 amino acids from the terminus) of theheme-binding molecule and/or composition. In some embodiments, theconjugation domain comprises a functional group for conjugating orlinking the heme-binding molecule and/or composition to the carrierscaffold. Some exemplary functional groups for conjugation include, butare not limited to, an amino group, a N-substituted amino group, acarboxyl group, a carbonyl group, an acid anhydride group, an aldehydegroup, a hydroxyl group, an epoxy group, a thiol, a disulfide group, analkenyl group, a hydrazine group, a hydrazide group, a semicarbazidegroup, a thiosemicarbazide group, one partner of a binding pair, anamide group, an aryl group, an ester group, an ether group, a glycidylgroup, a halo group, a hydride group, an isocyanate group, an ureagroup, an urethane group, and any combinations thereof.

Activation agents can be used to activate the components to beconjugated together. Without limitations, any process and/or reagentknown in the art for conjugation activation can be used. Exemplaryactivation methods or reagents include, but are not limited to,1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC orEDAC), hydroxybenzotriazole (HOBT), N-Hydroxysuccinimide (NHS),2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (HATU), silanization, surfaceactivation through plasma treatment, and the like.

In some embodiments, the conjugation domain can comprise at least oneamino group that can be non-convalently or covalently coupled withfunctional groups on the carrier scaffold. For example, the primaryamines of the amino acid residues (e.g., lysine or cysteine residues)can be used to conjugate the heme-binding molecule and/or compositionwith the carrier scaffold. In some embodiments, the amino group at theN-terminus of the heme-binding molecules and/or compositions can be usedfor conjugating the heme-binding molecules and/or compositions with thecarrier scaffold.

Without limitations, the engineered heme-binding molecules and/orcompositions can be conjugated to the carrier-scaffold through covalentor non-covalent interactions or any combination of covalent andnon-covalent interactions. Further, conjugation can be accomplished anyof method known to those of skill in the art. For example, covalentimmobilization can be accomplished through, for example, silanecoupling. See, e.g., Weetall, 15 Adv. Mol. Cell Bio. 161 (2008);Weetall, 44 Meths. Enzymol. 134 (1976). The covalent interaction betweenthe engineered heme-binding molecules and/or compositions and/orcoupling molecule and the surface can also be mediated by otherart-recognized chemical reactions, such as NHS reaction or a conjugationagent. The non-covalent interaction between the engineered heme-bindingmolecules and/or compositions and/or coupling molecule and the surfacecan be formed based on ionic interactions, van der Waals interactions,dipole-dipole interactions, hydrogen bonds, electrostatic interactions,and/or shape recognition interactions.

Without limitations, conjugation can include either a stable or a labile(e.g. cleavable) bond or conjugation agent. Exemplary conjugationsinclude, but are not limited to, covalent bond, amide bond, additions tocarbon-carbon multiple bonds, azide alkyne Huisgen cycloaddition,Diels-Alder reaction, disulfide linkage, ester bond, Michael additions,silane bond, urethane, nucleophilic ring opening reactions: epoxides,non-aldol carbonyl chemistry, cycloaddition reactions: 1,3-dipolarcycloaddition, temperature sensitive, radiation (IR, near-IR, UV)sensitive bond or conjugation agent, pH-sensitive bond or conjugationagent, non-covalent bonds (e.g., ionic charge complex formation,hydrogen bonding, pi-pi interactions, hist guest interactions, such ascyclodextrin/adamantly host guest interaction) and the like.

In some embodiments, the heme-binding molecules and/or compositions canbe conjugated to the carrier-scaffold with a linker. In someembodiments, the heme-binding molecules and/or compositions can beconjugated to the carrier-scaffold with a linking group selected fromthe group consisting of a direct bond, an atom such as oxygen or sulfur,C(O), C(O)O, OC(O)O, C(O)NH, NHC(O)O, NH, SS, SO, SO₂, SO₃, and SO₂NH.

In some embodiments, the engineered heme-binding molecules and/orcompositions can be conjugated to the carrier scaffold by a couplingmolecule pair. The terms “coupling molecule pair” and “coupling pair” asused interchangeably herein refer to the first and second molecules thatspecifically bind to each other. One member of the binding pair isconjugated with the carrier scaffold while the second member isconjugated with the heme-binding molecules and/or compositions. As usedherein, the phrase “first and second molecules that specifically bind toeach other” refers to binding of the first member of the coupling pairto the second member of the coupling pair with greater affinity andspecificity than to other molecules. Exemplary coupling molecule pairsinclude, without limitations, any haptenic or antigenic compound incombination with a corresponding antibody or binding portion or fragmentthereof (e.g., digoxigenin and anti-digoxigenin; mouse immunoglobulinand goat antimouse immunoglobulin) and nonimmunological binding pairs(e.g., biotin-avidin, biotin-streptavidin), hormone (e.g., thyroxine andcortisol-hormone binding protein), receptor-receptor agonist,receptor-receptor antagonist (e.g., acetylcholine receptor-acetylcholineor an analog thereof), IgG-protein A, lectin-carbohydrate, enzyme-enzymecofactor, enzyme-enzyme inhibitor, and complementary oligonucleotidepairs capable of forming nucleic acid duplexes). The coupling moleculepair can also include a first molecule that is negatively charged and asecond molecule that is positively charged.

One example of using coupling pair conjugation is the biotin-avidin orbiotin-streptavidin conjugation. In this approach, one of the members ofmolecules to be conjugated together (e.g., the engineered heme-bindingmolecule and/or composition or the carrier scaffold) is biotinylated andthe other is conjugated with avidin or streptavidin. Many commercialkits are available for biotinylating molecules, such as proteins. Forexample, an aminooxy-biotin (AOB) can be used to covalently attachbiotin to a molecule with an aldehyde or ketone group. In someembodiments, AOB is attached to the engineered heme-binding moleculeand/or composition. Further, as described elsewhere herein, an AKTsequence on the N-terminal of the engineered heme-binding moleculesand/or compositions can allow the engineered heme-binding moleculesand/or compositions to be biotinylated at a single site and furtherconjugated to the streptavidin-coated solid surface. Moreover, theheme-binding molecule and/or composition can be coupled to a biotinacceptor peptide, for example, the AviTag or Acceptor Peptide (referredto as AP; Chen et al., 2 Nat. Methods 99 (2005)). The Acceptor Peptidesequence allows site-specific biotinylation by the E. coli enzyme biotinligase (BirA; Id.). Thus, in some embodiments, the conjugation domaincomprises an amino acid sequence of a biotin acceptor peptide.

Another non-limiting example of using conjugation with a couplingmolecule pair is the biotin-sandwich method. See, e.g., Davis et al.,103 PNAS 8155 (2006). In this approach, the two molecules to beconjugated together are biotinylated and then conjugated together usingtetravalent streptavidin. Another example for conjugation would be touse PLP-mediated bioconjugation. See, e.g., Witus et al., 132 JACS 16812(2010). Still another example of using coupling pair conjugation isdouble-stranded nucleic acid conjugation.

In this approach, one of the members of molecules to be conjugatedtogether is conjugated with a first strand of the double-strandednucleic acid and the other is conjugated with the second strand of thedouble-stranded nucleic acid. Nucleic acids can include, withoutlimitation, defined sequence segments and sequences comprisingnucleotides, ribonucleotides, deoxyribonucleotides, nucleotide analogs,modified nucleotides and nucleotides comprising backbone modifications,branchpoints and nonnucleotide residues, groups or bridges.

The carrier scaffold can also be functionalized to include a functionalgroup for conjugating with the heme-binding molecules and/orcompositions. In some embodiments, the carrier scaffold can befunctionalized to include a coupling molecule, or a functional fragmentthereof, that is capable of selectively binding with an engineeredheme-binding molecules and/or compositions described herein. As usedherein, the term “coupling molecule” refers to any molecule or anyfunctional group that is capable of selectively binding with anengineered microbe surface-binding domain described herein.Representative examples of coupling molecules include, but are notlimited to, antibodies, antigens, lectins, proteins, peptides, nucleicacids (DNA, RNA, PNA and nucleic acids that are mixtures thereof or thatinclude nucleotide derivatives or analogs); receptor molecules, such asthe insulin receptor; ligands for receptors (e.g., insulin for theinsulin receptor); and biological, chemical or other molecules that haveaffinity for another molecule.

In some embodiments, the coupling molecule is an aptamer. As usedherein, the term “aptamer” means a single-stranded, partiallysingle-stranded, partially double-stranded or double-stranded nucleotidesequence capable of specifically recognizing a selectednon-oligonucleotide molecule or group of molecules by a mechanism otherthan Watson-Crick base pairing or triplex formation. Aptamers caninclude, without limitation, defined sequence segments and sequencescomprising nucleotides, ribonucleotides, deoxyribonucleotides,nucleotide analogs, modified nucleotides and nucleotides comprisingbackbone modifications, branchpoints and nonnucleotide residues, groupsor bridges. Methods for selecting aptamers for binding to a molecule arewidely known in the art and easily accessible to one of ordinary skillin the art. The aptamers can be of any length, e.g., from about 1nucleotide to about 100 nucleotides, from about 5 nucleotides to about50 nucleotides, or from about 10 nucleotides to about 25 nucleotides.

In some embodiments, the heme-binding composition and/or molecule canfurther comprise a therapeutic agent. For example, the heme-bindingcomposition and/or molecule can comprise an anti-microbial agent.Therapeutic agents are described herein below. Any method available tothe skilled artisan for conjugating a therapeutic agent to a peptide canbe used for conjugating the therapeutic agent to the heme-bindingcomposition and/or molecule. For example, functional groups or methodsused for conjugating the molecule to a carrier scaffold can also be usedfor conjugating the molecule to a therapeutic agent. This can bebeneficial for delivering or concentrating a therapeutic agent (e.g., ananti-microbial agent) at a nidus of infection.

The multiple domains of a heme-binding molecule and/or composition canbe arranged in any desired orientation in the engineered heme-bindingmolecule and/or composition. For example, N-terminus of the heme-bindingdomain can be linked to the C-terminus of a second domain or C-terminusof the heme-binding domain can be linked to the N-terminus of a seconddomain. In some embodiments, that linking between the first and seconddomain is via the linker.

Further, as disclosed herein, an engineered heme-binding moleculesand/or compositions can comprise at least one heme-binding domain,including at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, at least tenor more heme-binding domains. When more than two first or second domainsare present, such domains can all be the same, all different, or somesame and some different.

In some embodiments, the engineered heme-binding molecule and/orcomposition disclosed herein comprises two or more heme-binding domainsand one second domain. In such molecules, one heme-binding domain can belinked to the second domain and the other heme-binding domains can belinked to the heme-binding domain linked to the second domain.Alternatively, two heme-binding domains can be linked to the seconddomain and other heme-binding domains can be linked to one or both ofthe two heme-binding domains linked to the second domain.

In some embodiments, the engineered heme-binding molecules and/orcompositions disclosed herein comprise two or more second domains andone heme-binding domain. In such molecules, one second domain can belinked to the heme-binding domain and the other second domains can belinked to the second domain linked to the heme-binding domain.Alternatively, two second domains can be linked to the heme-bindingdomain and other second domains can be linked to one or both of the twosecond domains linked to the heme-binding domain.

In some embodiments, the engineered heme-binding molecule and/orcomposition is in the form of a multimeric complex comprising at leasttwo (e.g., two, three, four, five, six, seven, eight, nine, ten, ormore) engineered heme-binding molecules and/or compositions.Accordingly, the multimeric complex can be a di-, tri-, tetra-, penta-,hexa- or higher multimeric complex. Without limitations, the multimericcomplex can be formed by interactions between a second domain or linkerof a first molecule with a second domain or a linker of the secondmolecule. Such interactions can comprise covalent linking ornon-covalent linking. The heme-binding molecules and/or compositions inthe multimeric complex can all be the same, all different, or some sameand some different.

In some embodiments, an engineered heme-binding molecule can furthercomprise a substrate binding domain. Non-limiting examples of substratebinding domains can include an Fc domain or AKT.

In some embodiments, a heme-binding composition and/or molecule asdescribed herein can further comprise a microbe-binding domain, e.g.conjugated to and/or in combination with molecules comprising ahemopexin domain. Non-limiting examples of microbe-binding domains caninclude MBL and CRP. The term “microbe binding domain” can refer to anymolecule or a fragment thereof that can specifically bind to the surfaceof a microbe or pathogen, e.g., any component present on a surface of amicrobe or pathogen, or any matter or component/fragment that isderived, originated or secreted from a microbe or pathogen. Moleculesthat can be used in the microbe binding domain can include, for example,but are not limited to, peptides, polypeptides, proteins,peptidomimetics, antibodies, antibody fragments (e.g., antigen bindingfragments of antibodies), carbohydrate-binding protein, e.g., a lectin,glycoproteins, glycoprotein-binding molecules, amino acids,carbohydrates (including mono-, di-, tri- and poly-saccharides), lipids,steroids, hormones, lipid-binding molecules, cofactors, nucleosides,nucleotides, nucleic acids (e.g., DNA or RNA, analogues and derivativesof nucleic acids, or aptamers), peptidoglycan, lipopolysaccharide, smallmolecules, and any combinations thereof.

Compositions and/or molecules comprising a microbe-binding domain can beused, e.g., for separating microbes from a test sample in vivo, in situor in vitro. Generally, the microbe-binding molecules disclosed hereincan bind with or capture at least one microbe. The microbe can be anintact or whole microbe or any matter or component that is derived,originated or secreted from a microbe. Any matter or component that isderived, originated or secreted from a microbe is also referred to as“microbial matter” herein. Thus, the microbe-binding molecules disclosedherein can bind/capture an intact or whole microbe or microbial matterderived, originated or secreted from the microbe. Exemplary microbialmatter that can bind to the microbe-binding molecule can include, but isnot limited to, a cell wall component, an outer membrane, a plasmamembrane, a ribosome, a microbial capsule, a pili or flagella, anyfragments of the aforementioned microbial components, any nucleic acid(e.g., DNA, including 16S ribosomal DNA, and RNA) derived from amicrobe, microbial endotoxin (e.g., lipopolysaccharide), and the like.In addition, microbial matter can encompass non-viable microbial matterthat can cause an adverse effect (e.g., toxicity) to a host or anenvironment. The terms “microbe-binding molecule(s)” and“microbe-targeting molecule(s)” are used interchangeably herein.

In accordance with the various embodiments described herein, moleculescan comprise at least one microbe-binding domain comprising at least aportion of a C-reactive protein (CRP) and at least one hemopexin domain.In some embodiments, the two domains can be conjugated together via alinker. In addition to the microbe-binding domain amino acid sequence,the molecule can further comprise one or more amino acids (e.g., one,two, three, four, five, six, seven, eight, nine, ten, or more) aminoacids on the N- or C-terminus of the microbe-binding sequence.Generally, the microbe-binding domain can have an amino acid sequence ofabout 10 to about 300 amino acid residues. In some embodiments, themicrobe-binding domain can have an amino acid sequence of about 50 toabout 250 amino acid residues. In some embodiments, the microbe-bindingdomain can have an amino acid sequence of at least about 5, at leastabout 10, at least about 15, at least about 20, at least about 30, atleast about 40, at least about 50, at least about 60, at least about 70,at least about 80, at least about 90, at least about 100 amino acidresidues or more. For any known sequences of a microbe-binding domain,one of skill in the art can determine the optimum length of amino acidsequence for retaining microbe-binding activity.

C-reactive protein (CRP) can bind with gram-positive microbe and can beused for capturing/detecting microbes. As used herein, “CRP” cancomprise full length CRP or a fragment thereof retaining microbe bindingactivity. Without limitations, the CRP can be from any source availableto one of skill in the art. For example, the CRP can be from a mammaliansource. For example, the CRP can be human CRP (NCBI Reference Sequence:NP_000558.2) or mouse CRP (NCBI Reference Sequence: NP_031794.3). Insome embodiments, the first domain comprises an amino acid sequencecomprising amino acids 19-224 of the human. CRP is described further inthe art, e.g., in U.S. Patent Application No. 61/917,705 filed Dec. 18,2013.

In some embodiments, the microbe-binding domain can comprise apeptidomimetic that mimics a molecule or a fragment thereof that canspecifically bind to the surface of a microbe or pathogen, or microbialmatter. For example, a microbe-binding domain can comprise apeptidomimetic that mimics a carbohydrate recognition domain or afragment thereof, e.g., carbohydrate recognition domain of MBL or afragment thereof.

In some embodiments, the microbe-binding domain can be a carbohydraterecognition domain or a fragment thereof of carbohydrate bindingprotein. The term “carbohydrate recognition domain” as used hereinrefers to a region, at least a portion of which, can bind tocarbohydrates on a surface of microbes or pathogens. In someembodiments, the second domain can comprise at least about 50% of thefull length CRD, including at least about 60%, at least about 70%, atleast about 80%, at least about 90% or higher, capable of binding tocarbohydrates on a microbe surface. In some embodiments, 100% of thecarbohydrate recognition domain can be used to bind to microbes orpathogens. In other embodiments, the carbohydrate recognition domain cancomprise additional regions that are not capable of carbohydratebinding, but can have other characteristics or perform other functions,e.g., to provide flexibility to the carbohydrate recognition domain wheninteracting with microbes or pathogens.

Exemplary carbohydrate-binding proteins include, but are not limited to,lectin, collectin, ficolin, mannose-binding lectin (MBL),maltose-binding protein, arabinose-binding protein, and glucose-bindingprotein. Additional carbohydrate-binding proteins that can be includedin the microbe-binding domain described herein can include, but are notlimited to, lectins or agglutinins that are derived from a plant, e.g.,Galanthus nivalis agglutinin (GNA) from the Galanthus (snowdrop) plant,and peanut lectin. In some embodiments, pentraxin family members (e.g.,C-reactive protein) can also be used as a carbohydrate-binding protein.Pentraxin family members can generally bind capsulated microbes. Withoutlimitation, the carbohydrate-binding proteins can be wild-type,recombinant or a fusion protein. The respective carbohydrate recognitiondomains for such carbohydrate-binding proteins are known in the art, andcan be modified for various embodiments of the engineeredmicrobe-binding molecules described herein.

Any art-recognized recombinant carbohydrate-binding proteins orcarbohydrate recognition domains can be used in the engineeredmolecules. For example, recombinant mannose-binding lectins, e.g., butnot limited to, the ones disclosed in the U.S. Pat. Nos. 5,270,199;6,846,649; U.S. Patent Application No. US 2004/0229212; and PCTApplication No. WO 2011/090954, filed Jan. 19, 2011, the contents of allof which are incorporated herein by reference, can be used inconstructing the molecules and compositions described herein.

In some embodiments, the CRD is from an MBL, a member of the collectinfamily of proteins. A native MBL is a multimeric structure (e.g., about650 kDa) composed of subunits, each of which contains three identicalpolypeptide chains. Each MBL polypeptide chain (containing 248 aminoacid residues in length with a signal sequence) comprises a N-terminalcysteine rich region, a collagen-like region, a neck region, and acarbohydrate recognition domain (CRD). The sequence of each region hasbeen identified and is well known in the art, e.g. human MBL isavailable in the NCBI BLAST database as accession number NP_000233. TheCRD of human MBL comprises amino acids 114 to 248 of NP_000233. In someembodiments, the carbohydrate recognition domain of the engineered MBLmolecule can comprise a fragment of the CRD. Exemplary amino acidsequences of such fragments include, but are not limited to, ND (SEQ IDNO: 29), EZN (SEQ ID NO: 30: where Z is any amino acid, e.g., P),NEGEPNNAGS (SEQ ID NO: 31) or a fragment thereof comprising EZN,GSDEDCVLL (SEQ ID NO: 32) or a fragment thereof comprising E, andLLLKNGQWNDVPCST (SEQ ID NO: 33) or a fragment thereof comprising ND.Modifications to such CRD fragments, e.g., by conservative substitution,are also within the scope described herein. In some embodiments, the MBLor a fragment thereof used in the engineered molecules described hereincan be a wild-type molecule or a recombinant molecule.

In some circumstances, complement or coagulation activation induced by acarbohydrate-binding protein or a fragment thereof can be undesirabledepending on various applications, e.g., in vivo administration fortreatment of sepsis. In such embodiments, the additional portion of thecarbohydrate-binding protein can exclude at least one of complement andcoagulation activation regions. By way of example, when thecarbohydrate-binding protein is mannose-binding lectin or a fragmentthereof, the mannose-binding lectin or a fragment thereof can exclude atleast one of the complement and coagulation activation regions locatedon the collagen-like region. In such embodiments, the mannose-bindinglectin or a fragment thereof can exclude at least about one amino acidresidue, including at least about two amino acid residues, at leastabout three amino acid residues, at least about four amino acidresidues, at least about five amino acid residues, at least about sixamino acid residues, at least about seven amino acid residues, at leastabout eight amino acid residues, at least about nine amino acidresidues, at least about ten amino acid residues or more, around aminoacid residue K55 or L56 of MBL (e.g. NCBI Ref Seq: NP_000233) Exemplaryamino sequences comprising K55 or L56 of MBL that can be excluded fromthe engineered molecule or compositions described herein include, butare not limited to, EPGQGLRGLQGPPGKLGPPGNPGPSGS (SEQ ID NO: 19), GKLG(SEQ ID NO: 20), GPPGKLGPPGN (SEQ ID NO: 21), RGLQGPPGKL (SEQ ID NO:22), GKLGPPGNPGPSGS (SEQ ID NO: 23), GLRGLQGPPGKLGPPGNPGP (SEQ ID NO:24), or any fragments thereof.

In some embodiments, the additional portion of the carbohydrate-bindingproteins can activate the complement system. In alternative embodiments,the additional portion of the carbohydrate-binding protein cannotactivate the complement system. In some embodiments, the additionalportion of the carbohydrate-binding protein can be selected, configured,or modified such that it does not activate the complement system.

In some embodiments, the microbe-binding domain can comprise a neckregion or a fragment thereof from a lectin. By neck region of a lectionis meant the portion of the lection than connects the CRD to rest of themolecule. Without wishing to be bound by theory, the neck region canprovide flexibility and proper orientation for binding to a microbesurface. When the microbe-binding molecule disclosed herein comprises aneck region and an additional second domain, the neck region can belocated between the first domain and the additional second domain, i.e.,the neck region can act as a linker for linking the first domain and theadditional second domain. In some embodiments, the second domain cancomprise one or more (e.g., one, two, three, four, five, six, seven,eight, nine, ten, or more) additional amino acids on the N- orC-terminus of the neck region. In some embodiments, the neck regioncomprises the amino acid sequence

(SEQ ID NO: 25) PDGDSSLAASERKALQTEMARIKKWLTFSLGKQ, (SEQ ID NO: 26)APDGDSSLAASERKALQTEMARIKKWLTFSLGKQ, (SEQ ID NO: 27) PDGDSSLAASERKALQTEMARIKKWLTFSLG, or (SEQ ID NO: 28)APDGDSSLAASERKALQTEMARIKKWLTFSLG.

Modifications to the microbe-binding domain, e.g., by conservativesubstitution, are also within the scope described herein. In someembodiments, the microbe-binding domain or a fragment thereof used inthe molecules described herein can be a wild-type molecule or arecombinant molecule.

In some embodiments, a molecule and/or composition as described hereincan further comprise a detectable label. As used herein, the term“detectable label” refers to a composition capable of producing adetectable signal indicative of the presence of a target. Detectablelabels include any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Suitable labels include fluorescent molecules,radioisotopes, nucleotide chromophores, enzymes, substrates,chemiluminescent moieties, bioluminescent moieties, and the like. Assuch, a label is any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means needed for the methods and devices described herein.

In some embodiments, the detectable label can be an imaging agent orcontrast agent. As used herein, the term “imaging agent” refers to anelement or functional group in a molecule that allows for the detection,imaging, and/or monitoring of the presence and/or progression of acondition(s), pathological disorder(s), and/or disease(s). The imagingagent can be an echogenic substance (either liquid or gas), non-metallicisotope, an optical reporter, a boron neutron absorber, a paramagneticmetal ion, a ferromagnetic metal, a gamma-emitting radioisotope, apositron-emitting radioisotope, or an x-ray absorber. As used herein theterm “contrast agent” refers to any molecule that changes the opticalproperties of tissue or organ containing the molecule. Opticalproperties that can be changed include, but are not limited to,absorbance, reflectance, fluorescence, birefringence, optical scatteringand the like. In some embodiments, the detectable labels also encompassany imaging agent (e.g., but not limited to, a bubble, a liposome, asphere, a contrast agent, or any detectable label described herein) thatcan facilitate imaging or visualization of a tissue or an organ in asubject, e.g., for diagnosis of an infection.

Suitable optical reporters include, but are not limited to, fluorescentreporters and chemiluminescent groups. A wide variety of fluorescentreporter dyes are known in the art. Typically, the fluorophore is anaromatic or heteroaromatic compound and can be a pyrene, anthracene,naphthalene, acridine, stilbene, indole, benzindole, oxazole, thiazole,benzothiazole, cyanine, carbocyanine, salicylate, anthranilate,coumarin, fluorescein, rhodamine or other like compound.

Exemplary fluorophores include, but are not limited to, 1,5 IAEDANS;1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein;5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein (pH 10);5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Carboxyfluorescein);5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA(5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE;7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD);7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ;Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine); AcridineOrange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin FeulgenSITSA; Aequorin (Photoprotein); Alexa Fluor350™; Alexa Fluor430™; AlexaFluor488™; Alexa Fluor 532™; Alexa Fluor546™; Alexa Fluor568™; AlexaFluor594™; Alexa Fluor 633™; Alexa Fluor647™; Alexa Fluor660™; AlexaFluor680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC,AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D;Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTS; AstrazonBrilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G;Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF(low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP(Y66H); BG-647; Bimane; Bisbenzamide; Blancophor FFG; Blancophor SV;BOBO™-1; BOBO™-3; Bodipy 492/515; Bodipy 493/503; Bodipy 500/510; Bodipy505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570;Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X;Bodipy 665/676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6GSE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR;Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™-1; BO-PRO™-3; BrilliantSulphoflavin FF; Calcein; Calcein Blue; Calcium Crimson™; Calcium Green;Calcium Green-1 Ca²⁺ Dye; Calcium Green-2 Ca²⁺; Calcium Green-5N Ca²⁺;Calcium Green-C18 Ca²⁺; Calcium Orange; Calcofluor White;Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow;Catecholamine; CFDA; CFP—Cyan Fluorescent Protein; Chlorophyll;Chromomycin A; Chromomycin A; CMFDA; Coelenterazine; Coelenterazine cp;Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazinehcp; Coelenterazine ip; Coelenterazine O; Coumarin Phalloidin; CPMMethylcoumarin; CTC; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™;Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); d2; Dabcyl; Dansyl;Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansylfluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH(Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123);Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di-16-ASP); DIDS;Dihydorhodamine 123 (DHR); DiO (DiOC18(3)); DiR; DiR (DiIC18(7));Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97;Eosin; Erythrosin; Erythrosin ITC; Ethidium homodimer-1 (EthD-1);Euchrysin; Europium (III) chloride; Europium; EYFP; Fast Blue; FDA;Feulgen (Pararosaniline); FITC; FL-645; Flazo Orange; Fluo-3; Fluo-4;Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold(Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™(high pH); Fura-2, high calcium; Fura-2, low calcium; Genacryl BrilliantRed B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow5GF; GFP (S65T); GFP red shifted (rsGFP); GFP wild type, non-UVexcitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv;Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst 33258; Hoechst33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine(FluoroGold); Hydroxytryptamine; Indodicarbocyanine (DiD);Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1;LaserPro; Laurodan; LDS 751; Leucophor PAF; Leucophor SF; Leucophor WS;Lissamine Rhodamine; Lissamine Rhodamine B; LOLO-1; LO-PRO-1; LuciferYellow; Mag Green; Magdala Red (Phloxin B); Magnesium Green; MagnesiumOrange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF;Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; MitotrackerGreen FM; Mitotracker Orange; Mitotracker Red; Mitramycin;Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS(Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red;Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow;Nylosan Brilliant lavin EBG; Oregon Green™; Oregon Green 488-X; OregonGreen™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue;Pararosaniline (Feulgen); PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5;PE-TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; PhorwiteBKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist;Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26; PKH67; PMIA;Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline;Procion Yellow; Propidium lodid (PI); PyMPO; Pyrene; Pyronine; PyronineB; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin;RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5GLD; Rhodamine 6G; Rhodamine B 540; Rhodamine B 200; Rhodamine B extra;Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine;Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal;R-phycoerythrin (PE); red shifted GFP (rsGFP, S65T); S65A; S65C; S65L;S65T; Sapphire GFP; Serotonin; Sevron Brilliant Red 2B; Sevron BrilliantRed 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™;sgBFP™ (super glow BFP); sgGFP™; sgGFP™ (super glow GFP); SITS; SITS(Primuline); SITS (Stilbene Isothiosulphonic Acid); SPQ(6-methoxy-N-(3-sulfopropyl)-quinolinium); Stilbene; Sulphorhodamine Bcan C; Sulphorhodamine G Extra; Tetracycline; Tetramethylrhodamine;Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); ThiazineRed R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN;Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR;TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC(TetramethylRodamineIsoThioCyanate); True Blue; TruRed; Ultralite;Uranine B; Uvitex SFC; wt GFP; WW 781; XL665; X-Rhodamine; XRITC; XyleneOrange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1;and YOYO-3. Many suitable forms of these fluorescent compounds areavailable and can be used.

Other exemplary detectable labels include luminescent and bioluminescentmarkers (e.g., biotin, luciferase (e.g., bacterial, firefly, clickbeetle and the like), luciferin, and aequorin), radiolabels (e.g., 3H,125I, 35S, 14C, or 32P), enzymes (e.g., galactosidases, glucorinidases,phosphatases (e.g., alkaline phosphatase), peroxidases (e.g.,horseradish peroxidase), and cholinesterases), and calorimetric labelssuch as colloidal gold or colored glass or plastic (e.g., polystyrene,polypropylene, and latex) beads. Patents teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345,4,277,437, 4,275,149, and 4,366,241, each of which is incorporatedherein by reference.

Suitable echogenic gases include, but are not limited to, a sulfurhexafluoride or perfluorocarbon gas, such as perfluoromethane,perfluoroethane, perfluoropropane, perfluorobutane,perfluorocyclobutane, perfluropentane, or perfluorohexane. Suitablenon-metallic isotopes include, but are not limited to, ¹¹C, ¹⁴C, ¹³N,¹⁸F, ¹²³I, ¹²⁴I, and ^(125I). Suitable radioisotopes include, but arenot limited to, ⁹⁹mTc, ⁹⁵Tc, ¹¹¹In, ⁶²Cu, Ga, ⁶⁸Ga, and ¹⁵³Gd. Suitableparamagnetic metal ions include, but are not limited to, Gd(III),Dy(III), Fe(III), and Mn(II). Suitable X-ray absorbers include, but arenot limited to, Re, Sm, Ho, Lu, Pm, Y, Bi, Pd, Gd, La, Au, Au, Yb, Dy,Cu, Rh, Ag, and Ir.

In some embodiments, the radionuclide is bound to a chelating agent orchelating agent-linker attached to the heme-binding molecule and/orcomposition. Suitable radionuclides for direct conjugation include,without limitation, ¹⁸F, ¹²⁴I, ¹²⁵I, ¹³¹I, and mixtures thereof.Suitable radionuclides for use with a chelating agent include, withoutlimitation, ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁸⁹Sr, ⁸⁶Y, ⁸⁷Y, ⁹⁰Y, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In,¹¹⁷mSn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, ²¹²Bi, andmixtures thereof. Suitable chelating agents include, but are not limitedto, DOTA, BAD, TETA, DTPA, EDTA, NTA, HDTA, their phosphonate analogs,and mixtures thereof. One of skill in the art will be familiar withmethods for attaching radionuclides, chelating agents, and chelatingagent-linkers to molecules such as the heme-binding molecule and/orcomposition and carrier scaffolds disclosed herein.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels can be detected using photographicfilm or scintillation counters, fluorescent markers can be detectedusing a photo-detector to detect emitted light. Enzymatic labels aretypically detected by providing the enzyme with an enzyme substrate anddetecting the reaction product produced by the action of the enzyme onthe enzyme substrate, and calorimetric labels can be detected byvisualizing the colored label. Exemplary methods for in vivo detectionor imaging of detectable labels include, but are not limited to,radiography, magnetic resonance imaging (MRI), Positron emissiontomography (PET), Single-photon emission computed tomography (SPECT, orless commonly, SPET), Scintigraphy, ultrasound, CAT scan, photoacousticimaging, thermography, linear tomography, poly tomography, zonography,orthopantomography (OPT or OPG), and computed Tomography (CT) orComputed Axial Tomography (CAT scan).

In some embodiments, the detectable label can include an enzyme.Exemplary enzymes for use as detectable labels include, but are notlimited to, horseradish peroxidase (HRP), alkaline phosphastase (AP), orany combinations thereof.

In some embodiments, the detectable can include an enzyme substrate(e.g., an microbial enzyme substrate) conjugated to a detectable agent.For example, the detectable agent can be any moiety that, when cleavedfrom an enzyme substrate by the enzyme, forms a detectable moiety butthat is not detectable in its conjugated state. The enzyme substrate,e.g. a microbial enzyme substrate can be a substrate specific for one ormore types of microbes to be detected, and it can be selected dependingupon what enzymes the microbe possesses or secretes. See, e.g.,International Patent Application: WO 2011/103144 for the use of suchdetectable label in detection of microbes, the content of which isincorporated herein by reference.

In some embodiments, the detectable label is a fluorophore or a quantumdot. Without wishing to be bound by a theory, using a fluorescentreagent can reduce signal-to-noise in the imaging/readout, thusmaintaining sensitivity. In some embodiments, the detectable label is agold particle.

In some embodiments, the detectable label can be configured to include a“smart label”, which is undetectable when conjugated to the heme-bindingmolecules and/or compositions, but produces a color change when releasedfrom the engineered molecules in the presence of an enzyme, e.g. amicrobial enzyme. Thus, when a microbe binds to the engineeredmolecules, the microbe releases enzymes that release the detectablelabel from the engineered molecules. An observation of a color changeindicates presence of the microbe in the sample. In some embodiments,the detectable label can be a chromogenic or fluorogenic microbe enzymesubstrate so that when a microbe binds to the engineeredmicrobe-targeting molecule, the enzyme that the microbe releases caninteract with the detectable label to induce a color change. Examples ofsuch microbe enzyme substrate can include, but are not limited to,indoxyl butyrate, indoxyl glucoside, esculin, magneta glucoside,red-β-glucuronide, 2-methoxy-4-(2-nitrovinyl) phenylβ-D-glu-copyranoside, 2-methoxy-4-(2-nitrovinyl) phenylβ-D-cetamindo-2-deoxyglucopyranoside, and any other art-recognizedmicrobe enzyme substrates. Such embodiments can act as an indicator forthe presence of a microbe or pathogen or enzyme.

In one aspect, described herein is a method of reducing the level offree heme in the blood of a subject, the method comprising contactingthe blood of the subject with a heme-binding composition as describedherein. In some embodiments, the method can comprise administering thecomposition to the subject. In some embodiments, the method can compriseremoving a portion of the subject's blood prior to the contacting stepand performing the contacting step extracorporeally and then returningthe portion of the subject's blood to the subject.

In some embodiments, the heme-binding compositions described herein,e.g. the compositions comprising the hemopexin polypeptides describedherein can bind to myoglobin.

In one aspect, described herein is a method of reducing the level offree myoglobin in the blood of a subject, the method comprisingcontacting the blood of the subject with a heme-binding composition asdescribed herein. In some embodiments, the method can compriseadministering the composition to the subject. In some embodiments, themethod can comprise removing a portion of the subject's blood prior tothe contacting step and performing the contacting step extracorporeallyand then returning the portion of the subject's blood to the subject.

In some embodiments, described herein is a method of treating, e.g.crush injury and/or rhabdomyolysis in a subject by administering aheme-binding molecule and/or composition as described herein to thesubject. Rhabdomyolysis can arise from a number of causes, e.g. crushinjury, infections, toxins, etc. and cause kidney damage. In someembodiments, administration can comprise contacting the blood of thesubject with the heme-binding molecule and/or composition. In someembodiments, the method can comprise administering the molecule and/orcomposition to the subject. In some embodiments, the method can compriseremoving a portion of the subject's blood prior to the contacting stepand performing the contacting step extracorporeally and then returningthe portion of the subject's blood to the subject.

In some embodiments, the extracorporeal device is a device as describedin, e.g. International Patent Publications WO2012/135834 andWO2011/091037; each of which is incorporated by reference herein in itsentirety. Further extracorporeal devices for blood filtration andmethods of constructing them are well known in the art, see, e.g.International Patent Publications PCT/US04/012911; PCT/US05/065126;PCT/USO4/040923; PCT/SE87/006471; PCT/IB11/056000; PCT/US90/006924;PCT/US06/0016747; PCT/JP10/072557; U.S. Patent Publications2011/0272343; 2012/0220915 and U.S. Pat. Nos. 3,954,623; 7,059,480;7,217,365; 7,014,648; 4,517,090; 7,488,302; 7,332,096; each of which isincorporated by reference herein in its entirety. By way of non-limitingexample, the device can comprise a blood removal means, e.g. a needleand attached tubing, a filtration unit, and a blood return means, e.g. asecond tubing and needle. The filtration unit can comprise a substratewith a large surface area, e.g. a filter, column, membrane, poroussurface, channels, and the like. Blood filtration devices can optionallyfurther comprise pumps, syringes, blood storage compartments,reservoirs, tubing, sterilization means, and the like.

In some embodiments, the extracorporeal device can comprise aheme-binding molecule and/or composition as described herein conjugatedto a hollow fiber DLT-like device, e.g. the HEMOPURIFIER™ device(Aethlon Medical; San Diego, Calif.). Further information can be foundin the art at, e.g., U.S. Pat. Nos. 6,254,567; 8,105,487; 6,117,100;U.S. Patent Publication 2012/0164628; International Patent PublicationWO2012135834; WO2006041125; WO2010065765; and European Patent No.2694970, 2344233; 1624785.

In one aspect, described herein is a composition comprising aheme-binding molecule or composition as described herein, and furthercomprising a solid substrate or support to which the heme-bindingmolecule or composition is conjugated. In some embodiments, the solidsubstrate or support can be a hollow fiber, e.g. the hollow fiber of aDLT device, as described above herein.

In some embodiments, the heme-binding composition is bound to a solidsubstrate of an extracorporeal device, e.g. a filter, affinity column,cavity or tube. Non-limiting examples of solid substrate include ahollow-fiber reactor or any other blood filtration membrane or flowdevice (e.g., a simple dialysis tube) or other resins, fibers, or sheetsto which the heme-binding composition can be bound. In some embodiments,binding can be non-covalent, e.g., by hydrogen, electrostatic, or vander waals interactions, however, binding may also be covalent. By“conjugated” is meant the covalent linkage of at least two molecules. Insome embodiments, the heme-binding composition can be conjugated to aprotein on the solid substrate.

In some embodiments, the heme-binding molecule and/or composition can bebound to, e.g. a bead or particle. The beards and/or particles can becontacted with the subject's blood. Heme present in the blood will bebound by the heme-binding molecule and/or composition and the complex ofheme and heme-binding molecule and/or composition can then be removedfrom the blood by, e.g. centrifugation to pellet the beads or applyingmagnetic field to separate magnetic beads from the blood. As usedherein, the term “bead” refers to a microparticle of any design orconstruction, but preferably a microparticle that is about the size of acell or smaller. While cell sizes vary according to cell type, the bead(microparticles) can be of any such size or smaller, e.g. nanoscale insize. In some embodiments, the beads or particles can range in size from1 nm to 1 mm. In some embodiments, the beads can be about 250 nm toabout 250 μm in size.

The bead can be formed of any material to which a heme-binding moleculeand/or composition can be bound. Suitable materials include, withoutlimitation, a synthetic polymer, biopolymer, latex, or silica, and thematerial may have paramagnetic properties. The use of such beads and/orparticles is known in the art and described, e.g. magnetic bead andnano-particles are well known and methods for their preparation havebeen described in the are art, for example in U.S. Pat. Nos. 6,878,445;5,543,158; 5,578,325; 6,676,729; 6,045,925 and 7,462,446, and U.S. Pat.Pub. Nos.: 2005/0025971; 2005/0200438; 2005/0201941; 2005/0271745;2006/0228551; 2006/0233712; 2007/01666232 and 2007/0264199, contents ofall of which are herein incorporated by reference in their entirety.Magnetic microbeads are easily and widely available commercially, withor without functional groups capable of binding to affinity molecules.Suitable superparamagnetic microbeads are commercially available such asfrom Dynal Inc. of Lake Success, N. Y.; PerSeptive Diagnostics, Inc. ofCambridge, Mass.; Invitrogen Corp. of Carlsbad, Calif.; Cortex BiochemInc. of San Leandro, Calif.; and Bangs Laboratories of Fishers, Ind.

In some embodiments, provided herein is an article or product fortargeting or binding microbes comprising at least one, including atleast two, at least three, at least four, at least five, at least ten,at least 25, at least 50, at least 100, at least 250, at least 500, ormore engineered heme-binding molecules and/or compositions conjugated toa carrier scaffold or a surface thereof. The “carrier scaffold” is alsoreferred to as a “carrier substrate” herein. In some embodiments,surface of the carrier scaffold can be coated with the heme-bindingmolecules and/or compositions disclosed herein. As used herein, the term“article” refers to any distinct physical microscale or macroscaleobject. An article comprising a heme-binding molecule and/or compositionconjugated to a carrier scaffold is also referred to as a “heme-bindingarticle” herein.

Without limitations, the carrier scaffold can be selected from a widevariety of materials and in a variety of formats. For example, thecarrier scaffold can be utilized in the form of beads or particles(including nanoparticles, microparticles, polymer microbeads, magneticmicrobeads, and the like), filters, fibers, screens, mesh, tubes, hollowfibers, scaffolds, plates, channels, gold particles, magnetic materials,planar shapes (such as a rectangular strip or a circular disk, or acurved surface such as a stick), other substrates commonly utilized inassay formats, and any combinations thereof.

Examples of carrier scaffolds include, but are not limited to, nucleicacid scaffolds, protein scaffolds, lipid scaffolds, dendrimers,microparticles or microbeads, nanotubes, microtiter plates, medicalapparatuses (e.g., needles or catheters) or implants, dipsticks or teststrips, microchips, filtration devices or membranes, membranes,diagnostic strips, hollow-fiber reactors, microfluidic devices, livingcells and biological tissues or organs, extracorporeal devices, mixingelements (e.g., spiral mixers), and the like. In some embodiments, thecarrier scaffold can be in the form of a continuous roll on which thetest area(s) and optionally reference area(s) are present in the form ofcontinuous lines or a series of spots.

The carrier scaffold can be made of any material, including, but notlimited to, metal, metal alloy, polymer, plastic, paper, glass, fabric,packaging material, biological material such as cells, tissues,hydrogels, proteins, peptides, nucleic acids, and any combinationsthereof.

In some embodiments, the heme-binding articles disclosed herein can beused to capture, detect, or remove heme and/or myoglobin from any sourceor in any fluid, e.g., a biological fluid (e.g., blood sample). In someembodiments where the fluid is blood, after removal of the heme and/ormyoglobin from the blood collected from a subject with the heme-bindingmagnetic microbeads, the blood can be circulated back to the samesubject as a therapeutic intervention. Alternatively, the carrierscaffold can comprise a hollow-fiber reactor or any other bloodfiltration membrane or flow device (e.g., a simple dialysis tube, spiralmixer or static mixer) or other resins, fibers, or sheets to selectivebind and sequester the heme and/or myoglobin.

The particular format or material of the carrier scaffold depends on theparticular use or application, for example, the separation/detectionmethods employed in an assay application. In some embodiments, theformat or material of the carrier scaffold can be chosen or modified tomaximize signal-to-noise ratios, e.g., to minimize background binding orfor ease of separation of reagents and cost. For example, carrierscaffold can be treated or modified with surface chemistry to minimizechemical agglutination and non-specific binding. In some embodiments, atleast a portion of the carrier scaffold surface that is in contact witha test sample can be treated to become less adhesive to any molecules(including microbes, if any) present in a test sample. By way of exampleonly, the carrier scaffold surface in contact with a test sample can besalinized or coated with a polymer such that the surface is inert to themolecules present in the test sample, including but not limited to,cells or fragments thereof (including blood cells and blood components),proteins, nucleic acids, peptides, small molecules, therapeutic agents,microbes, microorganisms and any combinations thereof. In otherembodiments, a carrier scaffold surface can be treated with anomniphobic layer. See, e.g., Wong T S et al., “Bioinspiredself-repairing slippery surfaces with pressure-stable omniphobicity.”(2011) Nature 477 (7365): 443-447, and International Application No.:PCT/US12/21928, the content of which is incorporated herein byreference, for methods to produce a slippery carrier scaffold surface.Accordingly, non-specific binding of molecules from the test sample to asubstrate surface can be reduced, thus increasing the sensitivity andspecificity of the heme-binding agent.

In some embodiments, the carrier scaffold can be fabricated from orcoated with a biocompatible material. As used herein, the term“biocompatible material” refers to any material that does notdeteriorate appreciably and does not induce a significant immuneresponse or deleterious tissue reaction, e.g., toxic reaction orsignificant irritation, over time when implanted into or placed adjacentto the biological tissue of a subject, or induce blood clotting orcoagulation when it comes in contact with blood. Suitable biocompatiblematerials include, for example, derivatives and copolymers ofpolyimides, poly(ethylene glycol), polyvinyl alcohol, polyethyleneimine,and polyvinylamine, polyacrylates, polyamides, polyesters,polycarbonates, and polystyrenes. In some embodiments, biocompatiblematerials can include metals, such as titanium and stainless steel, orany biocompatible metal used in medical implants. In some embodiments,biocompatible materials can include paper substrate, e.g., as a carrierscaffold for a diagnostic strip. In some embodiments, biocompatiblematerials can include peptides or nucleic acid molecules, e.g., anucleic acid scaffold such as a 2-D DNA sheet or 3-D DNA scaffold.

Additional material that can be used to fabricate or coat a carrierscaffold include, without limitations, polydimethylsiloxane, polyimide,polyethylene terephthalate, polymethylmethacrylate, polyurethane,polyvinylchloride, polystyrene polysulfone, polycarbonate,polymethylpentene, polypropylene, polyvinylidine fluoride, polysilicon,polytetrafluoroethylene, polysulfone, acrylonitrile butadiene styrene,polyacrylonitrile, polybutadiene, poly(butylene terephthalate),poly(ether sulfone), poly(ether ether ketones), poly(ethylene glycol),styrene-acrylonitrile resin, poly(trimethylene terephthalate), polyvinylbutyral, polyvinylidenedifluoride, poly(vinyl pyrrolidone), and anycombination thereof.

In some embodiments, the carrier scaffold can be fabricated from orcoated with a biodegradable material. As used herein, the term“biodegradable” refers to the ability of a composition to erode ordegrade in vivo to form smaller chemical fragments. Degradation canoccur, for example, by enzymatic, chemical or physical processes.Non-limiting examples of biodegradable polymers that can be used inaspects provided herein include poly(lactide)s, poly(glycolide)s,poly(lactic acid)s, poly(glycolic acid)s, poly (lactide-co-glycolide),polyanhydrides, polyorthoesters, polycaprolactone, polyesteramides,polycarbonate, polycyanoacrylate, polyurethanes, polyacrylate, blendsand copolymers thereof.

Other additional biodegradable polymers include biodegradablepolyetherester copolymers. Generally speaking, the polyetherestercopolymers are amphiphilic block copolymers that include hydrophilic(for example, a polyalkylene glycol, such as polyethylene glycol) andhydrophobic blocks (for example, polyethylene terephthalate). Anexemplary block copolymer is, but is not limited to, poly(ethyleneglycol)-based and poly(butylene terephthalate)-based blocks (PEG/PBTpolymer). PEG/PBT polymers are commercially available from OctoPlus Inc,under the trade designation PolyActive™. Non-limiting examples ofbiodegradable copolymers or multiblock copolymers include the onesdescribed in U.S. Pat. Nos. 5,980,948 and 5,252,701, the contents ofwhich are incorporated herein by reference.

Other biodegradable polymer materials include biodegradableterephthalate copolymers that include a phosphorus-containing linkage.Polymers having phosphoester linkages, called poly(phosphates),poly(phosphonates) and poly(phosphites), are known in the art. See, forexample, Penczek et al., Handbook of Polymer Synthesis, Chapter 17:“Phosphorus-Containing Polymers,” 1077-1 132 (Hans R. Kricheldorf ed.,1992), as well as U.S. Pat. Nos. 6,153,212; 6,485,737; 6,322,797;6,600,010; 6,419,709; 6,419,709; 6,485,737; 6,153,212; 6,322,797 and6,600,010, the contents of which are incorporated herein by reference.

Biodegradable polyhydric alcohol esters can also be used as a materialof a carrier scaffold (e.g., a microparticle) (See U.S. Pat. No.6,592,895, which is incorporated herein by reference). In someembodiments, the biodegradable polymer can be a three-dimensionalcrosslinked polymer network containing hydrophobic and hydrophiliccomponents which forms a hydrogel with a crosslinked polymer structure,such as the one described in U.S. Pat. No. 6,583,219. In yet furtherembodiments, the biodegradable polymer can comprise a polymer based uponα-amino acids (such as elastomeric copolyester amides or copolyesterurethanes, as described in U.S. Pat. No. 6,503,538, which isincorporated herein by reference).

In some embodiments, the carrier scaffold can comprise a paper,nitrocellulose, glass, plastic, polymer, membrane material, nylon, andany combinations thereof. This is useful for using the article as a teststrip of a dipstick.

As used herein, by the “coating” or “coated” is generally meant a layerof molecules or material formed on an outermost or exposed layer of asurface. With respect to a coating of engineered heme-binding moleculesand/or compositions on a carrier scaffold, the term “coating” or“coated” refers to a layer of engineered heme-binding molecules and/orcompositions formed on an outermost or exposed layer of a carrierscaffold surface. In some embodiments, the carrier scaffold surface canencompass an outer surface or an inner surface, e.g., with respect to ahollow structure.

The amount of the engineered heme-binding molecules and/or compositionsconjugated to or coating on a carrier scaffold can vary with a number offactors such as a surface area, conjugation/coating density, types ofengineered heme-binding molecules and/or compositions, and/or bindingperformance. A skilled artisan can determine the optimum density ofengineered heme-binding molecules and/or compositions on a carrierscaffold using any methods known in the art. By way of example only, formagnetic microparticles as a carrier scaffold, the amount of theengineered heme-binding molecules and/or compositions used forconjugating to or coating magnetic microparticles can vary from about 1wt % to about 30 wt %, or from about 5 wt % to about 20 wt %. In someembodiments, the amount of the engineered heme-binding molecules and/orcompositions used for conjugating to or coating magnetic microparticlescan be higher or lower, depending on a specific need. However, it shouldbe noted that if the amount of the engineered heme-binding moleculesand/or compositions used for conjugating to or coating the magneticmicroparticles is too low, the magnetic microparticles can show a lowerbinding performance with heme and/or myoglobin. On the contrary, if theamount of the engineered heme-binding molecules and/or compositions usedfor conjugating to or coating the magnetic microparticles is too high,the dense layer of the engineered heme-binding molecules and/orcompositions can exert an adverse influence on the magnetic propertiesof the magnetic microbeads, which in turn can degrade the efficiency ofseparating the magnetic microbeads from a fluid utilizing the magneticfield gradient. Similar concerns apply to other substrate types.

In some embodiments, the carrier scaffold can further comprise at leastone area adapted for use as a reference area. By way of example only,the reference area can be adapted for use as a positive control,negative control, a reference, or any combination thereof. In someembodiments, the carrier scaffold can further comprise at least twoareas, wherein one area is adapted for a positive control and the secondarea is adapted for a negative control.

In some embodiments, the carrier scaffold can further comprise at leastone reference area or control area for comparison with a readout signaldetermined from the test area. The reference area generally excludes theengineered heme-binding molecules and/or compositions, e.g., to accountfor any background signal. In some embodiments, the reference area caninclude one or more known amounts of the detectable label that theengineered heme-binding molecules and/or compositions in the test areaencompass. In such embodiments, the reference area can be used forcalibration such that the amount of heme and/or myoglobin in a testsample can be estimated or quantified.

In some embodiments, the carrier scaffold can further comprise adetectable label. The detectable label can be separate from theheme-binding molecules and/or compositions conjugated with the carrierscaffold or linked to the heme-binding molecules and/or compositionsconjugated with the carrier scaffold.

Heme-binding microparticles: In some embodiments, the carrier scaffoldis a microparticle. Accordingly, some embodiments described hereinprovide a heme-binding microparticle comprising at least one engineeredheme-binding molecules and/or compositions on its surface. The term“microparticle” as used herein refers to a particle having a particlesize of about 0.001 μm to about 1000 μm, about 0.005 μm to about 50 μm,about 0.01 μm to about 25 μm, about 0.05 μm to about 10 μm, or about0.05 μm to about 5 μm. In one embodiment, the microparticle has aparticle size of about 0.05 μm to about 1 μm. In one embodiment, themicroparticle is about 0.09 μm-about 0.2 μm in size.

In some embodiments, the microparticle can range in size from 1 nm to 1mm, about 2.5 nm to about 500 μm, or about 5 nm to about 250 μm in size.In some embodiments, microparticle can be about 5 nm to about 100 μm insize. In some embodiments, microparticle can be about 0.01 μm to about10 μm in size. In some embodiments, the microparticle can be about 0.05μm to about 5 μm in size. In some embodiments, the microparticle can beabout 0.08 μm to about 1 μm in size. In one embodiment, themicroparticle can be about 10 nm to about 10 μm in size. In someembodiments, the the microparticle can be about 1 nm to about 1000 nm,from about 10 nm to about 500 nm, from about 25 nm to about 300 nm, fromabout 40 nm to about 250 nm, or from about 50 nm to about 200 nm. In oneembodiment, the microparticle can be about 50 nm to about 200 nm.

It will be understood by one of ordinary skill in the art thatmicroparticles usually exhibit a distribution of particle sizes aroundthe indicated “size.” Unless otherwise stated, the term “size” as usedherein refers to the mode of a size distribution of microparticles,i.e., the value that occurs most frequently in the size distribution.Methods for measuring the microparticle size are known to a skilledartisan, e.g., by dynamic light scattering (such as photocorrelationspectroscopy, laser diffraction, low-angle laser light scattering(LALLS), and medium-angle laser light scattering (MALLS)), lightobscuration methods (such as Coulter analysis method), or othertechniques (such as rheology, and light or electron microscopy).

Without limitations, the microparticle can be of any shape. Thus, themicroparticle can be, but is not limited to, spherical, rod, elliptical,cylindrical, disc, and the like. In some embodiments, the term“microparticle” as used herein can encompass a microsphere. The term“microsphere” as used herein refers to a microparticle having asubstantially spherical form. A substantially spherical microparticle isa microparticle with a difference between the smallest radii and thelargest radii generally not greater than about 40% of the smaller radii,and more typically less than about 30%, or less than 20%.

In some embodiments, the microparticles having a substantially sphericalshape and defined surface chemistry can be used to minimize chemicalagglutination and non-specific binding.

In one embodiment, the term “microparticle” as used herein encompasses amicrocapsule. The term “microcapsule” as used herein refers to amicroscopic capsule that contains an active ingredient, e.g., atherapeutic agent or an imagining agent. Accordingly, in someembodiments, the microparticles comprising on their surface engineeredheme-binding molecules and/or compositions can encapsulate at least oneactive ingredient therein, e.g., a therapeutic agent.

In general, any biocompatible material well known in the art forfabrication of microparticles can be used in embodiments of themicroparticle described herein. Accordingly, a microparticle comprisinga lipidic microparticle core is also within the scope described herein.An exemplary lipidic microparticle core is, but is not limited to, aliposome. A liposome is generally defined as a particle comprising oneor more lipid bilayers enclosing an interior, e.g., an aqueous interior.In one embodiment, a liposome can be a vesicle formed by a bilayer lipidmembrane. Methods for the preparation of liposomes are well described inthe art, e.g., Szoka and Papahadjopoulos (1980) Ann. Rev. Biophys.Bioeng. 9: 467, Deamer and Uster (1983) Pp. 27-51 In: Liposomes, ed. M.J. Ostro, Marcel Dekker, New York.

Heme-binding magnetic microparticles: In some embodiments, themicroparticle is a magnetic microparticle. Thus, in some embodiments,provided herein is a “heme-binding magnetic microparticle” wherein amagnetic microparticle comprising on its surface at least one engineeredheme-binding molecule and/or composition. Without limitations, suchheme-binding magnetic microparticles can be used to separate heme and/ormyoglobin from a test sample, e.g., but not limited to, any fluid,including a biological fluid such as blood. In some embodiments, theheme-binding magnetic microparticle can be used to remove heme and/ormyoglobin. Using magnetic microparticles as a substrate can beadvantageous because the heme-bound magnetic microparticles can beeasily separated from a sample fluid using a magnetic field gradient, beexamined for the presence of the heme and/or myoglobin. Thus, in someembodiments, the heme-binding magnetic microparticles can be used tocapture, detect, or remove heme and/or myoglobin contaminants from anysource or in any fluid, e.g., a biological fluid (e.g., blood sample).In some embodiments where the fluid is blood, after removal of the hemeand/or my from the blood collected from a subject with the heme-bindingmagnetic microbeads, the blood can be circulated back to the samesubject as a therapeutic intervention. Alternatively, the solidsubstrate can comprise a hollow-fiber reactor or any other bloodfiltration membrane or flow device (e.g., a simple dialysis tube, spiralmixer or static mixer) or other resins, fibers, or sheets to selectivebind and sequester heme and/or myoglobin.

Magnetic microparticles can be manipulated using magnetic field ormagnetic field gradient. Such particles commonly consist of magneticelements such as iron, nickel and cobalt and their oxide compounds.Magnetic microparticles are well-known and methods for their preparationhave been described in the art. See, e.g., U.S. Pat. Nos. 6,878,445;5,543,158; 5,578,325; 6,676,729; 6,045,925; and 7,462,446; and U.S.Patent Publications No. 2005/0025971; No. 2005/0200438; No.2005/0201941; No. 2005/0271745; No. 2006/0228551; No. 2006/0233712; No.2007/01666232; and No. 2007/0264199, the contents of which areincorporated herein by reference.

Magnetic microparticles are also widely and commercially available, withor without functional groups capable of conjugation with theheme-binding molecules and/or compositions disclosed herein. Magneticmicroparticles functionalized with various functional groups, e.g.,amino groups, carboxylic acid groups, epoxy groups, tosyl groups, orsilica-like groups, are also widely and commercially available. Suitablemagnetic microparticles are commercially available such as fromAdemTech, Miltenyi, PerSeptive Diagnostics, Inc. (Cambridge, Mass.);Invitrogen Corp. (Carlsbad, Calif.); Cortex Biochem Inc. (San Leandro,Calif.); and Bangs Laboratories (Fishers, Ind.). In particularembodiments, magnetic microparticles that can be used herein can be anyDYNABEADS® magnetic microbeads (Invitrogen Inc.), depending on thesubstrate surface chemistry.

Heme-binding microtiter plates: In some embodiments, the bottom surfaceof microtiter wells can be coated with the engineered heme-bindingmolecules and/or compositions described herein, e.g., for detectingand/or determining the amount of heme and/or myoglobin in a sample.After heme and/or myoglobin in the sample binding to the engineeredheme-binding molecules and/or compositions bound to the microwellsurface, the rest of the sample can be removed. Detectable moleculesthat can also bind to heme and/or myoglobin (e.g., an engineeredheme-binding molecules and/or compositions conjugated to a detectablemolecule as described herein) can then be added to the microwells withfor detection of heme and/or myoglobin. Various signal detection methodsfor determining the amount of proteins, e.g., using enzyme-linkedimmunosorbent assay (ELISA), with different detectable molecules havebeen well established in the art, and those signal detection methods canalso be employed herein to facilitate detection of the signal induced byheme and/or myoglobin binding on the engineered heme-binding moleculesand/or compositions.

Heme-binding dipsticks/test strips: In some embodiments, the carrierscaffold having the heme-binding molecules and/or compositionsconjugated thereon can be in the form of a dipstick and/or a test stripfor capture, detection, or clearance of heme and/or myoglobin. Forexample, a dipstick and/or a test strip can include at least one testarea containing one or more engineered heme-binding molecules and/orcompositions described herein. The dipstick and/or a test strip can bein any shape and/or in any format, e.g., a planar shape such as arectangular strip or a circular disk, or a curved surface such as astick. Alternatively, a continuous roll can be utilized, rather thandiscrete test strips, on which the test area(s) and optionally referencearea(s) are present in the form of continuous lines or a series ofspots. In some embodiments, the heme-binding dipsticks or test stripsdescribed herein can be used as point-of-care diagnostic tools for hemeand/or myoglobin.

In some embodiments, the carrier scaffold in the form of a dipstick or atest strip can be made of any material, including, without limitations,paper, nitrocellulose, glass, plastic, polymer, membrane material,nylon, and any combinations thereof. In one embodiment, the carrierscaffold in the form of a dipstick or a test strip can include paper. Inone embodiment, the carrier scaffold in the form of a dipstick or a teststrip can include nylon.

In some embodiments, the dipstick or a test strip can further compriseat least one reference area or control area for comparison with areadout signal determined from the test area. The reference areagenerally excludes the engineered heme-binding molecules and/orcompositions, e.g., to account for any background signal. In someembodiments, the reference area can include one or more known amounts ofthe detectable label that the engineered heme-binding molecules and/orcompositions in the test area encompass. In such embodiments, thereference area can be used for calibration such that the amount of hemeand/or myoglobin in a test sample can be estimated or quantified.

In some embodiments, the dipstick/test strip can further comprise adetectable label as described herein. The detectable label can be linkedto the heme-binding molecule conjugated with the dipstick/test strip orseparate from the heme-binding molecule conjugated with thedipstick/test strip.

In one embodiment, about 1 μg to about 100 μg heme-binding molecules canbe coated on or attached to a dipstick or membrane surface. In anotherembodiment, about 3 μg to about 60 μg heme-binding molecules can becoated on or attached to a dipstick or membrane surface. In someembodiments, about 0.1 mg/mL to about 50 mg/mL, about 0.5 mg/mL to about40 mg/mL, about 1 mg/mL to about 30 mg/mL, about 5 mg/mL to about 20mg/mL heme-binding molecules and/or compositions can be coated on orattached to a dipstick or membrane surface.

In one aspect, described herein is a method of producing a heme-bindingmolecule and/or composition, the method comprising culturing a cellcomprising a nucleic acid, e.g. an isolated nucleic acid, encoding aheme-binding molecule and/or composition as described herein underconditions suitable for the production of proteins and purifying theheme-binding molecule and/or composition by affinity purification with aFc domain binding reagent.

A nucleic acid encoding a heme-binding molecule and/or composition canbe a nucleic acid encoding, e.g. SEQ ID NO: 4 or SEQ ID NO: 5. Nucleicacid molecules encoding a heme-binding molecule and/or compositiondescribed herein are prepared by a variety of methods known in the art.These methods include, but are not limited to, PCT, ligation, and directsynthesis. A nucleic acid sequence encoding a polypeptide as describedherein can be recombined with vector DNA in accordance with conventionaltechniques, including blunt-ended or staggered-ended termini forligation, restriction enzyme digestion to provide appropriate termini,filling in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and ligation with appropriateligases. Techniques for such manipulations are disclosed, e.g., byManiatis et al., Molecular Cloning, Lab. Manual (Cold Spring Harbor Lab.Press, N Y, 1982 and 1989), and Ausubel, 1987, 1993, and can be used toconstruct nucleic acid sequences which encode a heme-binding moleculeand/or composition polypeptide as described herein.

The term “vector” encompasses any genetic element that is capable ofreplication when associated with the proper control elements and thatcan transfer gene sequences to cells. A vector can include, but is notlimited to, a cloning vector, an expression vector, a plasmid, phage,transposon, cosmid, chromosome, virus, virion, etc. These transgenes canbe introduced as a linear construct, a circular plasmid, or a viralvector, which can be an integrating or non-integrating vector. Thetransgene can also be constructed to permit it to be inherited as anextrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA(1995) 92:1292).

In one aspect, the technology described herein relates to an expressionvector comprising a nucleic acid encoding any of the heme-bindingmolecule and/or composition polypeptides described herein. Such vectorscan be used, e.g. to transform a cell in order to produce the encodedpolypeptide. As used herein, the term “expression vector” refers to avector that directs expression of an RNA or polypeptide from sequenceslinked to transcriptional regulatory sequences on the vector. Thesequences expressed will often, but not necessarily, be heterologous tothe cell. An expression vector may comprise additional elements, forexample, the expression vector may have two replication systems, thusallowing it to be maintained in two organisms, for example in mammaliancells for expression and in a prokaryotic host for cloning andamplification. The term “expression” refers to the cellular processesinvolved in producing RNA and proteins and as appropriate, secretingproteins, including where applicable, but not limited to, for example,transcription, transcript processing, translation and protein folding,modification and processing. “Expression products” include RNAtranscribed from a gene, and polypeptides obtained by translation ofmRNA transcribed from a gene. The term “gene” means the nucleic acidsequence which is transcribed (DNA) to RNA in vitro or in vivo whenoperably linked to appropriate regulatory sequences. The gene may or maynot include regions preceding and following the coding region, e.g. 5′untranslated (5′UTR) or “leader” sequences and 3′ UTR or “trailer”sequences, as well as intervening sequences (introns) between individualcoding segments (exons).

By “recombinant vector” is meant a vector that includes a heterologousnucleic acid sequence, or “transgene” that is capable of expression invivo. It should be understood that the vectors described herein can, insome embodiments, be combined with other suitable compositions andtherapies. Vectors useful for the delivery of a sequence encoding anisolated peptide as described herein can include one or more regulatoryelements (e.g., promoter, enhancer, etc.) sufficient for expression ofthe isolated peptide in the desired target cell or tissue. Theregulatory elements can be chosen to provide either constitutive orregulated/inducible expression. As used herein, the term “viral vector”refers to a nucleic acid vector construct that includes at least oneelement of viral origin and has the capacity to be packaged into a viralvector particle. The viral vector can contain the nucleic acid encodingan antibody or antigen-binding portion thereof as described herein inplace of non-essential viral genes. The vector and/or particle may beutilized for the purpose of transferring any nucleic acids into cellseither in vitro or in vivo. Numerous forms of viral vectors are known inthe art.

Examples of vectors useful in delivery of nucleic acids encodingisolated peptides as described herein include plasmid vectors, non-viralplasmid vectors (e.g. see U.S. Pat. Nos. 6,413,942, 6,214,804,5,580,859, 5,589,466, 5,763,270 and 5,693,622, all of which areincorporated herein by reference in their entireties); retroviruses(e.g. see U.S. Pat. No. 5,219,740; Miller and Rosman (1989)BioTechniques 7:980-90; Miller, A. D. (1990) Human Gene Therapy 1:5-14;Scarpa et al. (1991) Virology 180:849-52; Miller et al., Meth. Enzymol.217:581-599 (1993); Burns et al. (1993) Proc. Natl. Acad. Sci. USA90:8033-37; Boris-Lawrie and Temin (1993) Curr. Opin. Genet. Develop.3:102-09. Boesen et al., Biotherapy 6:291-302 (1994); Clowes et al., J.Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994);Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossmanand Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993), thecontents of each of which are herein incorporated by reference in theirentireties); lentiviruses (e.g., see U.S. Pat. Nos. 6,143,520;5,665,557; and 5,981,276, the contents of which are herein incorporatedby reference in their entireties; adenovirus-based expression vectors(e.g., see Haj-Ahmad and Graham (1986) J. Virol. 57:267-74; Bett et al.(1993) J. Virol. 67:5911-21; Mittereder et al. (1994) Human Gene Therapy5:717-29; Seth et al. (1994) J. Virol. 68:933-40; Barr et al. (1994)Gene Therapy 1:51-58; Berkner, K. L. (1988) BioTechniques 6:616-29; andRich et al. (1993) Human Gene Therapy 4:461-76; Wu et al. (2001)Anesthes. 94:1119-32; Parks (2000) Clin. Genet. 58:1-11; Tsai et al.(2000) Curr. Opin. Mol. Ther. 2:515-23; and U.S. Pat. Nos. 6,048,551;6,306,652 and 6,306,652, incorporated herein by reference in theirentireties); Adeno-associated viruses (AAV) (e.g. see U.S. Pat. Nos.5,139,941; 5,622,856; 5,139,941; 6,001,650; and 6,004,797, the contentsof each of which are incorporated by reference herein in theirentireties); and avipox vectors (e.g. see WO 91/12882; WO 89/03429; andWO 92/03545; which are incorporated by reference herein in theirentireties).

Useful methods of transfection can include, but are not limited toelectroporation, sonoporation, protoplast fusion, peptoid delivery, ormicroinjection. See, e.g., Sambrook et al (1989) Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratories, New York, for adiscussion of techniques for transforming cells of interest; andFelgner, P. L. (1990) Advanced Drug Delivery Reviews 5:163-87, for areview of delivery systems useful for gene transfer. Exemplary methodsof delivering DNA using electroporation are described in U.S. Pat. Nos.6,132,419; 6,451,002, 6,418,341, 6,233,483, U.S. Patent Publication No.2002/0146831, and International Publication No. WO/0045823, all of whichare incorporated herein by reference in their entireties.

Non-limiting examples of vectors useful for expression in prokaryoticcells can include plasmids. Plasmid vectors can include, but are notlimited to, pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18,pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK +/− orKS+/− (see “Stratagene Cloning Systems” Catalog (1993) from Stratagene,La Jolla, Calif, which is hereby incorporated by reference), pQE,pIH821, pGEX, pET series (see Studier et. al., “Use of T7 RNA Polymeraseto Direct Expression of Cloned Genes,” Gene Expression Technology, vol.185 (1990), which is hereby incorporated by reference in its entirety).Non-limiting examples of mammalian and insect appropriate vectors caninclude pcDNA3, pCMV6, pOptiVec, pFUSE, and pFastBac.

In some embodiments, the polypeptide can be constitutively expressed. Insome embodiments, nucleic acids encoding the polypeptide can beoperatively linked to a constitutive promoter. In some embodiments, thepolypeptide can be inducibly expressed. In some embodiments, nucleicacids encoding the polypeptide can be operatively linked to an induciblepromoter. As described herein, an “inducible promoter” is one that ischaracterized by initiating or enhancing transcriptional activity whenin the presence of, influenced by, or contacted by an inducer orinducing agent than when not in the presence of, under the influence of,or in contact with the inducer or inducing agent. An “inducer” or“inducing agent” may be endogenous, or a normally exogenous compound orprotein that is administered in such a way as to be active in inducingtranscriptional activity from the inducible promoter. In someembodiments, the inducer or inducing agent, e.g., a chemical, a compoundor a protein, can itself be the result of transcription or expression ofa nucleic acid sequence (e.g., an inducer can be a transcriptionalrepressor protein), which itself may be under the control or aninducible promoter. Non-limiting examples of inducible promoters includebut are not limited to, the lac operon promoter, a nitrogen-sensitivepromoter, an IPTG-inducible promoter, a salt-inducible promoter, andtetracycline, steroid-responsive promoters, rapamycin responsivepromoters and the like. Inducible promoters for use in prokaryoticsystems are well known in the art, see, e.g. the beta.-lactamase andlactose promoter systems (Chang et al., Nature, 275: 615 (1978, which isincorporated herein by reference); Goeddel et al., Nature, 281: 544(1979), which is incorporated herein by reference), the arabinosepromoter system, including the araBAD promoter (Guzman et al., J.Bacteriol., 174: 7716-7728 (1992), which is incorporated herein byreference; Guzman et al., J. Bacteriol., 177: 4121-4130 (1995), which isincorporated herein by reference; Siegele and Hu, Proc. Natl. Acad. Sci.USA, 94: 8168-8172 (1997), which is incorporated herein by reference),the rhamnose promoter (Haldimann et al., J. Bacteriol., 180: 1277-1286(1998), which is incorporated herein by reference), the alkalinephosphatase promoter, a tryptophan (trp) promoter system (Goeddel,Nucleic Acids Res., 8: 4057 (1980), which is incorporated herein byreference), the PLtetO-1 and Plac/are-1 promoters (Lutz and Bujard,Nucleic Acids Res., 25: 1203-1210 (1997), which is incorporated hereinby reference), and hybrid promoters such as the tac promoter. deBoer etal., Proc. Natl. Acad. Sci. USA, 80: 21-25 (1983), which is incorporatedherein by reference. Non-limiting examples of mammalian and insectpromoters can include CMV, SV40, LTR, and polyhedrin promoter.

An inducible promoter useful in the methods and systems as disclosedherein can be induced by one or more physiological conditions, such aschanges in pH, temperature, radiation, osmotic pressure, salinegradients, cell surface binding, and the concentration of one or moreextrinsic or intrinsic inducing agents. The extrinsic inducer orinducing agent may comprise amino acids and amino acid analogs,saccharides and polysaccharides, nucleic acids, protein transcriptionalactivators and repressors, cytokines, toxins, petroleum-based compounds,metal containing compounds, salts, ions, enzyme substrate analogs,hormones, and combinations thereof. In specific embodiments, theinducible promoter is activated or repressed in response to a change ofan environmental condition, such as the change in concentration of achemical, metal, temperature, radiation, nutrient or change in pH. Thus,an inducible promoter useful in the methods and systems as disclosedherein can be a phage inducible promoter, nutrient inducible promoter,temperature inducible promoter, radiation inducible promoter, metalinducible promoter, hormone inducible promoter, steroid induciblepromoter, and/or hybrids and combinations thereof. Appropriateenvironmental inducers can include, but are not limited to, exposure toheat (i.e., thermal pulses or constant heat exposure), various steroidalcompounds, divalent cations (including Cu2+ and Zn2+), galactose,tetracycline, IPTG (isopropyl-β-D thiogalactoside), as well as othernaturally occurring and synthetic inducing agents and gratuitousinducers.

Inducible promoters useful in the methods and systems as disclosedherein also include those that are repressed by “transcriptionalrepressors” that are subject to inactivation by the action ofenvironmental, external agents, or the product of another gene. Suchinducible promoters may also be termed “repressible promoters” where itis required to distinguish between other types of promoters in a givenmodule or component of the biological switch converters describedherein. Preferred repressors for use in the present invention aresensitive to inactivation by physiologically benign agent. Thus, where alac repressor protein is used to control the expression of a promotersequence that has been engineered to contain a lacO operator sequence,treatment of the host cell with IPTG will cause the dissociation of thelac repressor from the engineered promoter containing a lacO operatorsequence and allow transcription to occur. Similarly, where a tetrepressor is used to control the expression of a promoter sequence thathas been engineered to contain a tetO operator sequence, treatment ofthe host cell with tetracycline will cause the dissociation of the tetrepressor from the engineered promoter and allow transcription of thesequence downstream of the engineered promoter to occur.

The cell comprising the nucleic acid can be, e.g. a microbial cell or amammalian cell. In some embodiments, the cell as described herein iscultured under conditions suitable for the expression of theheme-binding composition polypeptide. Such conditions can include, butare not limited to, conditions under which the cell is capable of growthand/or polypeptide synthesis. Conditions may vary depending upon thespecies and strain of cell selected. Conditions for the culture ofcells, e.g. prokaryotic and mammalian cells, are well known in the art.If the recombinant polypeptide is operatively linked to an induciblepromoter, such conditions can include the presence of the suitableinducing molecule(s).

As used herein, “a Fc domain binding reagent” refers to an agent that iscapable of binding specifically to a Fc domain. In some embodiments, aFc domain binding reagent can be an anti-Fc antibody or a FcR receptoror portion thereof. The term “agent” refers generally to any entitywhich is normally not present or not present at the levels beingadministered to a cell. An agent can be selected from a groupcomprising: polynucleotides; polypeptides; small molecules; antibodies;or functional fragments thereof. As used herein, the term “specificbinding” refers to a chemical interaction between two molecules,compounds, cells and/or particles wherein the first entity binds to thesecond, target entity with greater specificity and affinity than itbinds to a third entity which is a non-target. In some embodiments,specific binding can refer to an affinity of the first entity for thesecond target entity which is at least 10 times, at least 50 times, atleast 100 times, at least 500 times, at least 1000 times or greater thanthe affinity for the third nontarget entity.

As used herein, “purifying” refers to the process of isolating aparticular molecule or composition and/or treating a sample comprising aparticular molecule or composition such that the molecule or compositionis more isolated than before the treatment (e.g. is present at a higherlevel of purity). The term “isolated” or “partially purified” as usedherein refers to a molecule or composition separated from at least oneother component (e.g., nucleic acid or polypeptide) that is present withthe molecule as found in its natural source and/or that would be presentwith the molecule when expressed by a cell, or secreted in the case ofsecreted polypeptides. A chemically synthesized nucleic acid orpolypeptide or one synthesized using in vitro transcription/translationis considered “isolated.”

In some embodiments, the polypeptides described herein can be purifyingby means of a agent specific for one or more domains of the polypeptide,e.g. a substrate and/or antibody reagent that binds specifically to,e.g., Fc, a linker, a microbe-binding domain, etc.

In some embodiments, the methods described herein relate to treating asubject having or diagnosed as having sepsis with a method orcomposition described herein. Subjects having sepsis can be identifiedby a physician using current methods of diagnosing sepsis. Symptomsand/or complications of sepsis which characterize these conditions andaid in diagnosis are well known in the art and include but are notlimited to, high fever, hot, flushed skin, elevated heart rate,hyperventilation, altered mental status, swelling, and low bloodpressure. Tests that may aid in a diagnosis of, e.g. sepsis include, butare not limited to, blood cultures. Exposure to risk factors for sepsis(e.g. immunodeficiency) can also aid in determining if a subject islikely to have sepsis or in making a diagnosis of sepsis.

In some embodiments, the methods described herein comprise administeringan effective amount of compositions described herein, e.g. aheme-binding molecule and/or composition to a subject in order toalleviate a symptom of sepsis and/or excess heme in the blood. As usedherein, “alleviating a symptom of sepsis” is ameliorating any conditionor symptom associated with the sepsis. As compared with an equivalentuntreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%,60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. Avariety of means for administering the compositions described herein tosubjects are known to those of skill in the art. Such methods caninclude, but are not limited to oral, parenteral, intravenous,intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary,injection, or cutaneous administration. Administration can be local orsystemic.

In some embodiments, the methods described herein can compriseadministering an effective amount of the compositions described herein,e.g. a heme-binding molecule and/or composition, to a subject in need oftreatment for rhabdomyolysis (e.g., crush injury).

The term “effective amount” as used herein refers to the amount of acomposition needed to alleviate at least one or more symptom of thedisease or disorder, and relates to a sufficient amount ofpharmacological composition to provide the desired effect. The term“therapeutically effective amount” therefore refers to an amount of acomposition that is sufficient to provide a particular effect whenadministered to a typical subject. An effective amount as used herein,in various contexts, would also include an amount sufficient to delaythe development of a symptom of the disease, alter the course of asymptom disease (for example but not limited to, slowing the progressionof a symptom of the disease), or reverse a symptom of the disease. Thus,it is not generally practicable to specify an exact “effective amount”.However, for any given case, an appropriate “effective amount” can bedetermined by one of ordinary skill in the art using only routineexperimentation.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dosage can vary depending upon the dosage formemployed and the route of administration utilized. The dose ratiobetween toxic and therapeutic effects is the therapeutic index and canbe expressed as the ratio LD50/ED50. Compositions and methods thatexhibit large therapeutic indices are preferred. A therapeuticallyeffective dose can be estimated initially from cell culture assays.Also, a dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC50 (i.e., theconcentration of the composition which achieves a half-maximalinhibition of symptoms) as determined in cell culture, or in anappropriate animal model. Levels in plasma can be measured, for example,by high performance liquid chromatography. The effects of any particulardosage can be monitored by a suitable bioassay, e.g., assay for thelevel of free heme in the blood of a subject, among others. The dosagecan be determined by a physician and adjusted, as necessary, to suitobserved effects of the treatment.

In some embodiments, the technology described herein relates to apharmaceutical composition comprising a heme-binding molecule and/orcomposition as described herein, and optionally a pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers and diluentsinclude saline, aqueous buffer solutions, solvents and/or dispersionmedia. The use of such carriers and diluents is well known in the art.Some non-limiting examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C₂-C₁₂ alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” or the like are used interchangeably herein. In someembodiments, the carrier inhibits the degradation of the active agent,e.g. the heme-binding composition as described herein.

In some embodiments, the pharmaceutical composition comprising aheme-binding molecule and/or composition as described herein can be aparenteral dose form. Since administration of parenteral dosage formstypically bypasses the patient's natural defenses against contaminants,parenteral dosage forms are preferably sterile or capable of beingsterilized prior to administration to a patient. Examples of parenteraldosage forms include, but are not limited to, solutions ready forinjection, dry products ready to be dissolved or suspended in apharmaceutically acceptable vehicle for injection, suspensions ready forinjection, and emulsions. In addition, controlled-release parenteraldosage forms can be prepared for administration of a patient, including,but not limited to, DUROS®-type dosage forms and dose-dumping.

Suitable vehicles that can be used to provide parenteral dosage forms ofa heme-binding molecule and/or composition as disclosed within are wellknown to those skilled in the art. Examples include, without limitation:sterile water; water for injection USP; saline solution; glucosesolution; aqueous vehicles such as but not limited to, sodium chlorideinjection, Ringer's injection, dextrose Injection, dextrose and sodiumchloride injection, and lactated Ringer's injection; water-misciblevehicles such as, but not limited to, ethyl alcohol, polyethyleneglycol, and propylene glycol; and non-aqueous vehicles such as, but notlimited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyloleate, isopropyl myristate, and benzyl benzoate. Compounds that alteror modify the solubility of a pharmaceutically acceptable salt can alsobe incorporated into the parenteral dosage forms of the disclosure,including conventional and controlled-release parenteral dosage forms.

The methods described herein can further comprise administering a secondagent and/or treatment to the subject, e.g. as part of a combinatorialtherapy. Non-limiting examples of a second agent and/or treatment caninclude antibiotics, fluid replacement, ultrafiltration, hemofiltration,dialysis, hemodialysis, hemodiafiltration, mechanical ventilation,insulin to control blood sugar levels, and vasopressors.

In some embodiments, treatment can comprise blood filtration of asubject in need of treatment for sepsis, as described above herein. Insome embodiments, the filtration is performed extracoporeally.

In certain embodiments, an effective dose of a composition comprising aheme-binding molecule and/or composition as described herein can beadministered to a patient, or the patient subjected to blood filtrationusing a heme-binding composition described herein, once. In certainembodiments, an effective dose of a composition comprising aheme-binding molecule and/or composition as described herein can beadministered to a patient, or the patient subjected to blood filtrationusing a heme-binding composition described herein, repeatedly. Forsystemic administration, subjects can be administered a therapeuticamount of a composition comprising a heme-binding molecule and/orcomposition, such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg,2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40mg/kg, 50 mg/kg, or more.

In some embodiments, after an initial treatment regimen, the treatmentscan be administered on a less frequent basis. For example, aftertreatment biweekly for three months, treatment can be repeated once permonth, for six months or a year or longer. Treatment according to themethods described herein can reduce levels of a marker or symptom of acondition, e.g. sepsis by at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80% or at least 90% or more.

The dosage of a composition as described herein can be determined by aphysician and adjusted, as necessary, to suit observed effects of thetreatment. With respect to duration and frequency of treatment, it istypical for skilled clinicians to monitor subjects in order to determinewhen the treatment is providing therapeutic benefit, and to determinewhether to increase or decrease dosage, increase or decreaseadministration frequency, discontinue treatment, resume treatment, ormake other alterations to the treatment regimen. The dosing schedule canvary from once a week to daily depending on a number of clinicalfactors, such as the subject's sensitivity to heme levels. The desireddose or amount of activation can be administered at one time or dividedinto subdoses, e.g., 2-4 subdoses and administered over a period oftime, e.g., at appropriate intervals through the day or otherappropriate schedule. In some embodiments, administration can bechronic, e.g., one or more doses and/or treatments daily over a periodof weeks or months. Examples of dosing and/or treatment schedules areadministration daily, twice daily, three times daily or four or moretimes daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month,2 months, 3 months, 4 months, 5 months, or 6 months, or more. Acomposition comprising a heme-binding molecule and/or composition can beadministered over a period of time, such as over a 5 minute, 10 minute,15 minute, 20 minute, or 25 minute period.

The dosage ranges for the administration of a heme-binding moleculeand/or composition according to the methods described herein dependupon, for example, the form of the polypeptide, its potency, and theextent to which symptoms, markers, or indicators of a conditiondescribed herein are desired to be reduced, for example the percentagereduction desired for free heme levels in the blood. The dosage shouldnot be so large as to cause adverse side effects. Generally, the dosagewill vary with the age, condition, and sex of the patient and can bedetermined by one of skill in the art. The dosage can also be adjustedby the individual physician in the event of any complication.

The efficacy of a heme-binding molecule and/or composition in, e.g. thetreatment of a condition described herein, or to induce a response asdescribed herein (e.g. a decrease in free heme levels in the blood) canbe determined by the skilled clinician. However, a treatment isconsidered “effective treatment,” as the term is used herein, if one ormore of the signs or symptoms of a condition described herein arealtered in a beneficial manner, other clinically accepted symptoms areimproved, or even ameliorated, or a desired response is induced e.g., byat least 10% following treatment according to the methods describedherein. Efficacy can be assessed, for example, by measuring a marker,indicator, symptom, and/or the incidence of a condition treatedaccording to the methods described herein or any other measurableparameter appropriate, e.g. the level of free heme in the blood.Efficacy can also be measured by a failure of an individual to worsen asassessed by hospitalization, or need for medical interventions (i.e.,progression of the disease is halted). Methods of measuring theseindicators are known to those of skill in the art and/or are describedherein. Treatment includes any treatment of a disease in an individualor an animal (some non-limiting examples include a human or an animal)and includes: (1) inhibiting the disease, e.g., preventing a worseningof symptoms (e.g. pain or inflammation); or (2) relieving the severityof the disease, e.g., causing regression of symptoms. An effectiveamount for the treatment of a disease means that amount which, whenadministered to a subject in need thereof, is sufficient to result ineffective treatment as that term is defined herein, for that disease.Efficacy of an agent can be determined by assessing physical indicatorsof a condition or desired response, (e.g. a decrease in free heme levelsin the blood). It is well within the ability of one skilled in the artto monitor efficacy of administration and/or treatment by measuring anyone of such parameters, or any combination of parameters. Efficacy canbe assessed in animal models of a condition described herein, forexample treatment of sepsis. When using an experimental animal model,efficacy of treatment is evidenced when a statistically significantchange in a marker is observed, e.g. the level of free heme in theblood.

In vitro assays are provided herein which allow the assessment of agiven dose of a composition. The efficacy of a given dosage combinationcan also be assessed in an animal model, e.g. an animal model of sepsis.

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed invention, because the scope of theinvention is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. If there is an apparent discrepancy between the usageof a term in the art and its definition provided herein, the definitionprovided within the specification shall prevail.

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here.

The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all usedherein to mean a decrease by a statistically significant amount. In someembodiments, “reduce,” “reduction” or “decrease” or “inhibit” typicallymeans a decrease by at least 10% as compared to a reference level (e.g.the absence of a given treatment) and can include, for example, adecrease by at least about 10%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or more. As used herein,“reduction” or “inhibition” does not encompass a complete inhibition orreduction as compared to a reference level. “Complete inhibition” is a100% inhibition as compared to a reference level. A decrease can bepreferably down to a level accepted as within the range of normal for anindividual without a given disorder.

The terms “increased”, “increase”, “enhance”, or “activate” are all usedherein to mean an increase by a statically significant amount. In someembodiments, the terms “increased”, “increase”, “enhance”, or “activate”can mean an increase of at least 10% as compared to a reference level,for example an increase of at least about 20%, or at least about 30%, orat least about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or up toand including a 100% increase or any increase between 10-100% ascompared to a reference level, or at least about a 2-fold, or at leastabout a 3-fold, or at least about a 4-fold, or at least about a 5-foldor at least about a 10-fold increase, or any increase between 2-fold and10-fold or greater as compared to a reference level. In the context of amarker or symptom, a “increase” is a statistically significant increasein such level.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Insome embodiments, the subject is a mammal, e.g., a primate, e.g., ahuman. The terms, “individual,” “patient” and “subject” are usedinterchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of sepsis.A subject can be male or female.

As used herein, “heme” refers to protoporhyrin IX (i.e. a compoundhaving the structure of Formula I) bound to Fe²⁺. In some embodiments,“heme” can additionally refer to hemin (i.e. the chloride salt ofprotoporphyrin IX-Fe³⁺) and/or hematin (i.e. protoporphyrin IX-Fe³⁺hydroxide).

As used herein, a “portion” refers to a part or fraction of a whole,e.g. a part or fraction of a total molecule. A particular molecule canhave multiple portions, e.g. two portions, three portions, fourportions, five portions, or more portions.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a condition in need of treatment(e.g. sepsis) or one or more complications related to such a condition,and optionally, have already undergone treatment for sepsis or the oneor more complications related to sepsis. Alternatively, a subject canalso be one who has not been previously diagnosed as having sepsis orone or more complications related to sepsis. For example, a subject canbe one who exhibits one or more risk factors for sepsis or one or morecomplications related to sepsis or a subject who does not exhibit riskfactors.

A “subject in need” of treatment for a particular condition can be asubject having that condition, diagnosed as having that condition, or atrisk of developing that condition.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably herein to designate a series of amino acid residues,connected to each other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and“polypeptide” refer to a polymer of amino acids, including modifiedamino acids (e.g., phosphorylated, glycated, glycosylated, etc.) andamino acid analogs, regardless of its size or function. “Protein” and“polypeptide” are often used in reference to relatively largepolypeptides, whereas the term “peptide” is often used in reference tosmall polypeptides, but usage of these terms in the art overlaps. Theterms “protein” and “polypeptide” are used interchangeably herein whenreferring to a gene product and fragments thereof. Thus, exemplarypolypeptides or proteins include gene products, naturally occurringproteins, homologs, orthologs, paralogs, fragments and otherequivalents, variants, fragments, and analogs of the foregoing.

As used herein, the term “nucleic acid” or “nucleic acid sequence”refers to any molecule, preferably a polymeric molecule, incorporatingunits of ribonucleic acid, deoxyribonucleic acid or an analog thereof.The nucleic acid can be either single-stranded or double-stranded. Asingle-stranded nucleic acid can be one nucleic acid strand of adenatured double-stranded DNA. Alternatively, it can be asingle-stranded nucleic acid not derived from any double-stranded DNA.In one aspect, the nucleic acid can be DNA. In another aspect, thenucleic acid can be RNA. Suitable nucleic acid molecules are DNA,including genomic DNA or cDNA. Other suitable nucleic acid molecules areRNA, including mRNA.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with a disease ordisorder, e.g. sepsis. The term “treating” includes reducing oralleviating at least one adverse effect or symptom of a condition,disease or disorder associated with sepsis. Treatment is generally“effective” if one or more symptoms or clinical markers are reduced.Alternatively, treatment is “effective” if the progression of a diseaseis reduced or halted. That is, “treatment” includes not just theimprovement of symptoms or markers, but also a cessation of, or at leastslowing of, progress or worsening of symptoms compared to what would beexpected in the absence of treatment. Beneficial or desired clinicalresults include, but are not limited to, alleviation of one or moresymptom(s), diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, remission (whetherpartial or total), and/or decreased mortality, whether detectable orundetectable. The term “treatment” of a disease also includes providingrelief from the symptoms or side-effects of the disease (includingpalliative treatment).

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carriere.g. a carrier commonly used in the pharmaceutical industry. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, the term “administering,” refers to the placement of acompound as disclosed herein into a subject by a method or route whichresults in at least partial delivery of the agent at a desired site.Pharmaceutical compositions comprising the compounds disclosed hereincan be administered by any appropriate route which results in aneffective treatment in the subject.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the method or composition, yet open to the inclusion ofunspecified elements, whether essential or not.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Definitions of common terms in cell biology and molecular biology can befound in “The Merck Manual of Diagnosis and Therapy”, 19th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); BenjaminLewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10:0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology:a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009,Wiley Intersciences, Coligan et al., eds.

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Sambrook et al.,Molecular Cloning: A Laboratory Manual (3 ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (2001); Davis et al.,Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc.,New York, USA (1995); Current Protocols in Protein Science (CPPS) (JohnE. Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocolsin Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley andSons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique byR. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal CellCulture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather andDavid Barnes editors, Academic Press, 1st edition, 1998) which are allincorporated by reference herein in their entireties.

Other terms are defined herein within the description of the variousaspects of the invention.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

The technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   -   1. An engineered heme-binding molecule comprising a hemopexin        domain and a second domain selected from the group consisting        of:        -   a linker; a microbe-binding molecule; and/or a substrate            binding domain;        -   wherein the second domain is conjugated to the hemopexin            domain.    -   2. The engineered heme-binding molecule of paragraph 1, wherein        the substrate binding domain is an Fc domain or AKT.    -   3. A heme-binding composition comprising a hemopexin domain        conjugated to an Fc domain.    -   4. The molecule or composition of any of paragraphs 1-3, further        comprising a detectable label.    -   5. A composition comprising the heme-binding molecule or        heme-binding composition of any of paragraphs 1-4 and further        comprising a microbe-binding domain.    -   6. The composition of paragraphs 1 or 5, wherein the        microbe-binding domain is selected from the group consisting of:        -   MBL and CRP.    -   7. The composition or molecule of any of paragraphs 1-6, further        comprising a solid substrate or support to which the        heme-binding molecule or composition is conjugated.    -   8. The composition or molecule of paragraph 7, wherein the solid        substrate or support is a hollow fiber.    -   9. The heme-binding composition or molecule of any of paragraphs        1-8, wherein the hemopexin domain is a polypeptide comprising        the sequence of SEQ ID NO: 2.    -   10. The heme-binding composition or molecule of any of        paragraphs 1-9, wherein the hemopexin domain is a polypeptide        having the sequence of SEQ ID NO: 2.    -   11. The heme-binding composition or molecule of any of        paragraphs 1-8, wherein the hemopexin domain comprises a        polypeptide having a sequence corresponding to residues 27-233        of SEQ ID NO: 2.    -   12. The heme-binding composition or molecule of any of        paragraphs 1-8 wherein the hemopexin domain comprises a        polypeptide having a sequence corresponding to residues 1-233 of        SEQ ID NO: 2.    -   13. The heme-binding composition or molecule of any of        paragraphs 1-8, wherein the hemopexin domain comprises a        polypeptide having a sequence corresponding to residues 27-220        of SEQ ID NO: 2.    -   14. The heme-binding composition or molecule of any of        paragraphs 1-8, wherein the hemopexin domain comprises a        polypeptide having a sequence corresponding to residues 1-220 of        SEQ ID NO: 2.    -   15. The heme-binding composition or molecule of any of        paragraphs 1-8, wherein the hemopexin domain comprises a        polypeptide having a sequence corresponding to residues 27-213        of SEQ ID NO: 2.    -   16. The heme-binding composition or molecule of any of        paragraphs 1-8, wherein the hemopexin domain comprises a        polypeptide having a sequence corresponding to residues 1-213 of        SEQ ID NO: 2.    -   17. The heme-binding composition or molecule of any of        paragraphs 1-16, wherein the hemopexin domain comprises a        mutation wherein the residues corresponding to residues 220-226        of SEQ ID NO: 2 have been replaced with a polypeptide linker of        about 1-10 amino acids in length.    -   18. The heme-binding composition or molecule of any of        paragraphs 1-17, wherein the hemopexin domain comprises a        mutation wherein the residues corresponding to residues 220-226        of SEQ ID NO: 2 have been replaced with the sequence GSGS (SEQ        ID NO: 18).    -   19. The heme-binding composition or molecule of any of        paragraphs 1-18, wherein the Fc domain is a polypeptide having        the sequence of SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 17.    -   20. A heme-binding composition of paragraph 3 having the        sequence of SEQ ID NO: 4 or SEQ ID NO: 5.    -   21. A method of reducing the level of free heme in the blood of        a subject, the method comprising contacting the blood of the        subject with the heme-binding composition or molecule of any of        paragraphs 1-20 or a molecule comprising a hemopexin domain.    -   22. A method of treating sepsis, the method comprising        administering an effective amount of a heme-binding composition        or molecule of any of paragraphs 1-20 or a molecule comprising a        hemopexin domain.    -   23. A method of reducing the level of myoglobin in the blood of        a subject, the method comprising contacting the blood of the        subject with the heme-binding composition or molecule of any of        paragraphs 1-20 or a molecule comprising a hemopexin domain.    -   24. A method of treating rhabdomyolysis or crush injury, the        method comprising administering an effective amount of a        heme-binding composition or molecule of any of paragraphs 1-20        or a molecule comprising a hemopexin domain.    -   25. The method of any of paragraphs 22 or 24, wherein the        administration comprises contacting the blood of the subject        with the heme-binding composition or molecule comprising a        hemopexin domain.    -   26. The method of any of paragraphs 21-25, further comprising        removing a portion of the subject's blood prior to the        contacting step and performing the contacting step        extracorporeally and then returning the portion of the subject's        blood to the subject.    -   27. The method of paragraph 26, wherein the heme-binding        composition or molecule comprising a hemopexin domain is bound        to a solid substrate of an extracorporeal device.    -   28. The method of paragraph 27, wherein the solid substrate is a        filter, affinity column, bear, or particle.    -   29. The method of any of paragraphs 21-28, wherein the molecule        comprising a hemopexin domain is a molecule consisting        essentially of a hemopexin domain.    -   30. The method of any of paragraphs 21-29, wherein the molecule        comprising a hemopexin domain is a molecule consisting of a        hemopexin domain.    -   31. The method of any of paragraphs 21-30, wherein the molecule        comprising a hemopexin domain has the sequence of any of SEQ ID        NOs: 1-2 or 9-16.    -   32. A method of producing a heme-binding composition or        molecule, the method comprising:        -   culturing a cell comprising a nucleic acid encoding a            heme-binding composition or molecule of any of paragraphs            1-20 under conditions suitable for the production of            proteins;        -   and purifying the heme-binding composition or molecule by            affinity purification with an stabilization domain binding            reagent, ion exchange purification, or size based            purification.    -   33. The method of paragraph 32, wherein the cell is selected        from the group consisting of:        -   a microbial cell; a mammalian cell; an insect cell; and a            plant cell.    -   34. A method of producing a heme-binding molecule or        composition, the method comprising:        -   maintaining a nucleic acid encoding a heme-binding            composition or molecule of any of paragraphs 1-20 under in            vitro transcription and/or in vitro translation conditions            suitable for the production of proteins;        -   and purifying the heme-binding composition by affinity            purification with an stabilization domain binding reagent,            ion exchange purification, or size based purification.

EXAMPLES Example 1: Fc Fusions to Hemopexin and Hemopexin Fragments forthe Treatment of Sepsis

Sepsis is a lethal condition that is often associated with a seriousmicrobial infection. However, while many hypotheses have been putforward, the exact cause of septic shock is not agreed upon andtherapeutics based on targeting the source of these various hypotheseshave generally failed in (or prior to) clinical trials. Studies haverecently suggested that excess free heme in the blood appears to play arole in the progression of sepsis and mechanism to remove the excessheme from blood could be very useful for patients suffering from sepsis.Host antimicrobial mechanisms reduce iron availability to pathogens.Iron proteins influencing the innate immune response include hepcidin,lactoferrin, siderocalin, haptoglobin, hemopexin, Nramp1, ferroportinand the transferrin receptor(1).

Under normal physiological conditions the protein hemopexin isresponsible for binding free heme and activating the liver to remove theexcess free heme from circulation. In a septic patient or animal,microbial infections can lead to a large increase in Red Blood Cell(RBS) lysis, which in turn leads to a significant increase in solublefree heme in the blood stream. This increase overwhelms the endogenouslevels of hemopexin leading to dangerously high levels of heme. Excessheme in the blood provides microbial pathogens with a readily availablesource of iron, which can be limiting agent in microbial growth andhemoglobin and heme may substantially contribute to microbe-inducedinflammation when bacterial or viral infection coexists with blood(2).In addition, free heme can have negative effects on an individual,although the exact mechanism has not been wholly determined.

At present there are no strategies to deal directly with high heme inthe blood. The current treatment generally includes administration ofantibiotics. Past clinical trials have focused on limiting the immunesystems response to microbial infections, thereby reducing the “CytokineStorm” that has been hypothesized to be the causative agent of sepsis.In addition, people have looked to use dialysis to remove solublecytokines—also to remove cytokines.

Described herein are Fc fusions to endogenous or engineered versions ofendogenous proteins to target heme for removal without introducing animmunogenic agent. Both full-length hemopexin and the amino terminaldomain of hemopexin have been shown to bind heme with binding constantsof 1 pM and 1 nM respectively. Described herein is the design andproduction of an Fc fusion to full-length human hemopexin, Fc fusions tomultiple fragments of the N-terminal domain of human hemopexin and an Fcfusion to full length hemopexin where the linker connecting the twostructural domains is replaced with a different polypeptide linker.

Expression and purification of recombinant versions of many endogenousproteins can be difficult and most experiments with hemopexin have usedhemopexin purified from blood(3).

Sequences of Fc-Hemopexin Fusions:

>aktFcHemopexin; A fusion protein of the following motifs listed fromN-terminus to C-terminus: the tripeptide Ala-Lys-Thr, the neck and Fcregion of human IgG1 (N297D), a single alanine insertion, humanhemopexin (with the leader sequence removed) (SEQ ID NO:3).

AKTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYDSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGATPLPPTSAHGNVAEGETKPDPDVTERCSDGWSFDATTLDDNGTMLFFKGEFVWKSHKWDRELISERWKNFPSPVDAAFRQGHNSVFLIKGDKVWVYPPEKKEKGYPKLLQDEFPGIPSPLDAAVECHRGECQAEGVLFFQGDREWFWDLATGTMKERSWPAVGNCSSALRWLGRYYCFQGNQFLRFDPVRGEVPPRYPRDVRDYFMPCPGRGHGHRNGTGHGNSTHHGPEYMRCSPHLVLSALTSDNHGATYAFSGTHYWRLDTSRDGWHSWPIAHQWPQGPSAVDAAFSWEEKLYLVQGTQVYVFLTKGGYTLVSGYPKRLEKEVGTPHGIILDSVDAAFICPGSSRLHIMAGRRLWWLDLKSGAQATWTELPWPHEKVDGALCMEKSLGPNSCSANGPGLYLIHGPNLYCYSDVEKLNAAKALPQPQNVTSLLGCTH>aktFcHemopexinNT: A fusion protein of the following motifs listed fromN-terminus to C-terminus: the tripeptide Ala-Lys-Thr, the neck and Fcregion of human IgG1 (N297D), a single alanine insertion, the N-terminaldomain of human hemopexin (residues 24-256 of the expressed protein)(SEQ ID NO: 4).

AKTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYDSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGATPLPPTSAHGNVAEGETKPDPDVTERCSDGWSFDATTLDDNGTMLFFKGEFVWKSHKWDRELISERWKNFPSPVDAAFRQGHNSVFLIKGDKVWVYPPEKKEKGYPKLLQDEFPGIPSPLDAAVECHRGECQAEGVLFFQGDREWFWDLATGTMKERSWPAVGNCSSALRWLGRYYCFQGNQFLRFDPVRGEVPPRYPRDVRDYFMPCPGRGHGHRNGTGHGNSTHHGPEYMR

The AKT tripeptide at the N-terminus of the Fc permits site-specificmodification of the protein and is optional. The N297D mutationgenerates an agylcosylated version of the Fc fragment, the wild typeasparagine (N297) can be used depending on the glycosylation statedesired for expression and Fc Receptor interactions.

Expression and Purification of Fc-Hemopexin fusions. The above geneswere cloned into a mammalian expression vector and transfected into 293Fcells (Invitrogen). Five days later the supernatant was collected andloaded onto a Protein A column (GE). Fc containing proteins bound toProtein A were eluted in low pH buffer and neutralized to pH 7. Theamount of purified protein was quantified and run on an SDS gel toconfirm its purity (Table 1 and FIG. 1).

TABLE 1 Yield (per liter Protein Predicted MW of cell culture)FcHemopexin 76 kDa 17 mg FcHemopexin-NT 53 kDa 30 mg FcHemopexin-G220 51kDa 15 mg FcHemopexin-H213 51 kDa 10 mg FcHemopexin-T24 73 kDa  1 mgHemopexin_G220H226GSGS 75 kDa  4 mg

Binding of Fc-Hemopexin fusions to free Hemin (Hemin is a chloride ionof heme). Fc-Hemopexin, Fc-Hemopexin-NT, FcHemopexin-G220,FcHemopexin-H213 and FcHemopexin-G220H226GSGS all bind free hemin andthe binding of hemin to Fc-Hemopexin is indistinguishable from heminbinding to native human Hemopexin (FIGS. 2, 4, and 5).

REFERENCES

-   1. E. E. Johnson, M. Wessling-Resnick, Iron metabolism and the    innate immune response to infection. Microbes and infection/Institut    Pasteur 14, 207 (March, 2012).-   2. T. Lin et al., Synergistic inflammation is induced by blood    degradation products with microbial Toll-like receptor agonists and    is blocked by hemopexin. The Journal of infectious diseases 202, 624    (Aug. 15, 2010).-   3. M. R. Mauk, A. Smith, A. G. Mauk, An alternative view of the    proposed alternative activities of hemopexin. Protein science: a    publication of the Protein Society 20, 791 (May, 2011).-   4. K. M. Lo et al., High level expression and secretion of Fc-X    fusion proteins in mammalian cells. Protein engineering 11, 495    (June, 1998).

Example 2

Different FcHx contructs' binding to Myoglobin were determined (Table2).

The polypeptides described in Table 2 were fused to the C-terminus ofSEQ ID NO: 17, which comprises the Fc fragment of human IgG with analanine-lysine-threonine tripeptide on the N-terminus and a singlealanine on the C-terminus. Table 2 summarizes the expression and bindingdata from these proteins.

The heme Binding of Fc fusions with variants of the N-terminal domain ofHemopexin was determined (FIG. 3). Free Heme was incubated with thespecified protein and then Free Heme was detected indirectly using anenzymatic heme dependent peroxidase reaction. Heme Binding of Fc Fusionswith variants of Full Length Hemopexin was also determined (FIG. 4).

FcHemopexin variants' binding to myoglobin was determined (FIG. 5).Myoglobin was coated in assay wells and then incubated with variousprotein probes, including the Fc Hemopexin fusions, the Fc alone(negative control), recombinant hemopexin and an anti-myoglobin antibody(positive control). The binding of the protein probe was then assayedusing a horse radish peroxidase detection system and the data for eachprotein probe was compared to wells coated with no Myoglobin.

TABLE 2 Starting Heme Myoglobin Fc fusion Protein SEQ ID NO: Reside*Last Residue* Expression Binding Binding Hemopexin 1 1 439 + + +HemopexinNT 9 1 233 + + + Hemopexin_T24 10 24 439 + NA NA Hemopexin_S2811 28 439 − NA NA HemopexinNT_G220 12 1 220 + + + HemopexinNT_H213 13 1213 + + + HemopexinNT_G212 14 1 212 − NA NA HemopexinNT_P207 15 1 207 −NA NA Hemopexin_mut3 16 1 439 + + + G220H226GSGS** *Starting and Endingresidues use the numbering system of mature human hemopexin **Residuesfrom gly 220 to thr 219 replaced with a gly-ser-gly-ser (SEQ ID NO: 18)linker

The general ELISA reagents and conditions were as follows: Wash buffer:PBS-T (175 ul×6); Incubation buffer: PBS; Pre-block=1% Milk and PBS (RTfor 1 hr); Incubation with Protein (RT for 1 hr); Antibody-HRPbuffer=0.5% Milk in PBS (RT for 1 hr).

What is claimed herein is:
 1. An engineered heme-binding moleculecomprising a heme-binding domain of human hemopexin and a human IgG Fcdomain; wherein the heme-binding domain is conjugated to the Fc domain;wherein the heme-binding domain comprises a polypeptide that is at least90% identical to a sequence corresponding to residues 27-213 of SEQ IDNO: 2; and wherein the heme-binding domain binds to heme.
 2. Theengineered heme-binding molecule of claim 1, wherein the heme-bindingdomain is at the N terminal of the Fc domain.
 3. The engineeredheme-binding molecule of claim 1, wherein the heme-binding domain is atthe C terminal of the Fc domain.
 4. The engineered heme-binding moleculeof claim 1, further comprising a detectable label.
 5. The engineeredheme-binding molecule of claim 1 further comprising a microbe-bindingdomain.
 6. The engineered heme-binding molecule of claim 5, wherein themicrobe-binding domain is selected from the group consisting of:mannose-binding lectin (MBL) and C-reactive protein (CRP).
 7. Theengineered heme-binding molecule of claim 1, wherein the heme-bindingdomain comprises a polypeptide that is at least 90% identical to asequence corresponding to: a) SEQ ID NO: 2; b) residues 27-233 of SEQ IDNO: 2; c) residues 1-233 of SEQ ID NO: 2; d) residues 27-220 of SEQ IDNO: 2; e) residues 1-220 of SEQ ID NO: 2; or f) residues 1-213 of SEQ IDNO:
 2. 8. The engineered heme-binding molecule of claim 1, wherein theheme-binding domain comprises a mutation wherein the residuescorresponding to residues 220-226 of SEQ ID NO: 2 have been replacedwith a polypeptide linker of about 1-10 amino acids in length.
 9. Theengineered heme-binding molecule of claim 1, wherein the heme-bindingdomain comprises a mutation wherein the residues corresponding toresidues 220-226 of SEQ ID NO: 2 have been replaced with the sequenceGSGS (SEQ ID NO: 18).
 10. The engineered heme-binding molecule of claim1, wherein the Fc domain is a polypeptide having the sequence of SEQ IDNO: 8, SEQ ID NO: 7 or SEQ ID NO:
 17. 11. The engineered heme-bindingmolecule of claim 1 having the sequence of SEQ ID NO: 3, SEQ ID NO: 4 orSEQ ID NO:
 5. 12. The engineered heme-binding molecule of claim 1,further comprising a linker or a substrate-binding domain.
 13. Theengineered heme-binding molecule of claim 12, wherein thesubstrate-binding domain comprises alanine-lysine-threonine (AKT) (SEQID NO: 35).
 14. The engineered heme-binding molecule of claim 1, whereinthe heme-binding domain comprises a polypeptide that is at least 90%identical to a sequence corresponding to: a. residues 24-462 of SEQ IDNO: 1; b. residues 24-256 of SEQ ID NO: 1; c. residues 27-233 of SEQ IDNO: 1; d. residues 1-233 of SEQ ID NO: 1; e. residues 27-220 of SEQ IDNO: 1; f. residues 1-220 of SEQ ID NO: 1; g. residues 27-213 of SEQ IDNO: 1; h. residues 1-213 of SEQ ID NO: 1; i. residues 27-256 of SEQ IDNO: 1; or j. residues 1-256 of SEQ ID NO:
 1. 15. The engineeredheme-binding molecule of claim 1, wherein the heme-binding domain bindsto heme and the Fc domain binds to an Fc receptor, thereby reducing thelevel of free heme or reducing the level of a molecule comprising hemein a solution.
 16. The engineered heme-binding molecule of claim 15,wherein the level of free heme or the level of the molecule comprisingheme is reduced by at least 20%.
 17. The engineered heme-bindingmolecule of claim 15, wherein the level of free heme or the level of themolecule comprising heme is reduced by at least 95%.
 18. The engineeredheme-binding molecule of claim 15, wherein the solution is blood.